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Sample records for systemic dystrophin splice

  1. Modulation of splicing of the preceding intron by antisense oligonucleotide complementary to intra-exon sequence deleted in dystrophin Kobe

    SciTech Connect

    Takeshima, Y.; Matuso, M.; Sakamoto, H.; Nishio, H.

    1994-09-01

    Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing a 52 bp deletion was skipped during splicing, although the known consensus sequences at the 5{prime} and 3{prime} splice site of exon 19 were maintained. These data suggest that the deleted sequence of exon 19 may function as a cis-acting factor for exact splicing for the upstream intron. To investigate this potential role, an in vitro splicing system using dystrophin precursors was established. A two-exon precursor containing exon 18, truncated intron 18, and exon 19 was accurately spliced. However, splicing of intron 18 was dramatically inhibited when wild exon 19 was replaced with mutated exon 19. Even though the length of exon 19 was restored to normal by replacing the deleted sequence with other sequence, splicing of intron 18 was not fully reactivated. Characteristically, splicing of intron 18 was inactivated more markedly when the replaced sequence contained less polypurine stretches. These data suggested that modification of the exon sequence would result in a splicing abnormality. Antisense 31 mer 2`-O-methyl ribonucleotide was targeted against 5{prime} end of deleted region of exon 19 to modulate splicing of the mRNA precursor. Splicing of intron 18 was inhibited in a dose- and time-dependent manner. This is the first in vitro evidence to show splicing of dystrophin pre-mRNA can be managed by antisense oligonucleotides. These experiments represent an approach in which antisense oligonucleotides are used to restore the function of a defective dystrophin gene in Duchenne muscular dystrophy by inducing skipping of certain exons during splicing.

  2. Electroporation Enhanced Effect of Dystrophin Splice Switching PNA Oligomers in Normal and Dystrophic Muscle.

    PubMed

    Brolin, Camilla; Shiraishi, Takehiko; Hojman, Pernille; Krag, Thomas O; Nielsen, Peter E; Gehl, Julie

    2015-01-01

    Peptide nucleic acid (PNA) is a synthetic DNA mimic that has shown potential for discovery of novel splice switching antisense drugs. However, in vivo cellular delivery has been a limiting factor for development, and only few successful studies have been reported. As a possible modality for improvement of in vivo cellular availability, we have investigated the effect of electrotransfer upon intramuscular (i.m.) PNA administration in vivo. Antisense PNA targeting exon 23 of the murine dystrophin gene was administered by i.m. injection to the tibialis anterior (TA) muscle of normal NMRI and dystrophic mdx mice with or without electroporation. At low, single PNA doses (1.5, 3, or 10 µg/TA), electroporation augmented the antisense exon skipping induced by an unmodified PNA by twofold to fourfold in healthy mouse muscle with optimized electric parameters, measured after 7 days. The PNA splice switching was detected at the RNA level up to 4 weeks after a single-dose treatment. In dystrophic muscles of the MDX mouse, electroporation increased the number of dystrophin-positive fibers about 2.5-fold at 2 weeks after a single PNA administration compared to injection only. In conclusion, we find that electroporation can enhance PNA antisense effects in muscle tissue. PMID:26623939

  3. Probabilistic simple splicing systems

    NASA Astrophysics Data System (ADS)

    Selvarajoo, Mathuri; Heng, Fong Wan; Sarmin, Nor Haniza; Turaev, Sherzod

    2014-06-01

    A splicing system, one of the early theoretical models for DNA computing was introduced by Head in 1987. Splicing systems are based on the splicing operation which, informally, cuts two strings of DNA molecules at the specific recognition sites and attaches the prefix of the first string to the suffix of the second string, and the prefix of the second string to the suffix of the first string, thus yielding the new strings. For a specific type of splicing systems, namely the simple splicing systems, the recognition sites are the same for both strings of DNA molecules. It is known that splicing systems with finite sets of axioms and splicing rules only generate regular languages. Hence, different types of restrictions have been considered for splicing systems in order to increase their computational power. Recently, probabilistic splicing systems have been introduced where the probabilities are initially associated with the axioms, and the probabilities of the generated strings are computed from the probabilities of the initial strings. In this paper, some properties of probabilistic simple splicing systems are investigated. We prove that probabilistic simple splicing systems can also increase the computational power of the splicing languages generated.

  4. Peptide Nucleic Acid Promotes Systemic Dystrophin Expression and Functional Rescue in Dystrophin-deficient mdx Mice

    PubMed Central

    Gao, Xianjun; Shen, Xiaoyong; Dong, Xue; Ran, Ning; Han, Gang; Cao, Limin; Gu, Ben; Yin, HaiFang

    2015-01-01

    Antisense oligonucleotide (AO)-mediated exon-skipping therapeutics shows great promise for Duchenne muscular dystrophy (DMD) patients. However, recent failure with drisapersen, an AO candidate drug in phase 3 trial, highlights the importance of exploring other effective AO chemistries for DMD. Previously, we demonstrated the appreciable biological activity of peptide nucleic acid (PNA) AOs in restoring dystrophin expression in dystrophin-deficient mdx mice intramuscularly. Here, we further explore the systemic potential and feasibility of PNA AOs in mediating exon skipping in mdx mice as a comprehensive systemic evaluation remains lacking. Systemic delivery of PNA AOs resulted in therapeutic level of dystrophin expression in body-wide peripheral muscles and improved dystrophic pathology in mdx mice without any detectable toxicity. Up to 40% of dystrophin restoration was achieved in gastrocnemius, to a less extent with other skeletal muscles, with no dystrophin in heart. Notably, comparable systemic activity was obtained between PNA AOs and phosphorodiamidate morpholino oligomer, a DMD AO chemistry in phase 3 clinical trial, under an identical dosing regimen. Overall, our data demonstrate that PNA is viable for DMD exon-skipping therapeutics with 20 mer showing the best combination of activity, solubility, and safety and further modifications to increase PNA aqueous solubility can enable longer, more effective therapeutics without the associated toxicity. PMID:26440599

  5. Peptide Nucleic Acid Promotes Systemic Dystrophin Expression and Functional Rescue in Dystrophin-deficient mdx Mice.

    PubMed

    Gao, Xianjun; Shen, Xiaoyong; Dong, Xue; Ran, Ning; Han, Gang; Cao, Limin; Gu, Ben; Yin, HaiFang

    2015-01-01

    Antisense oligonucleotide (AO)-mediated exon-skipping therapeutics shows great promise for Duchenne muscular dystrophy (DMD) patients. However, recent failure with drisapersen, an AO candidate drug in phase 3 trial, highlights the importance of exploring other effective AO chemistries for DMD. Previously, we demonstrated the appreciable biological activity of peptide nucleic acid (PNA) AOs in restoring dystrophin expression in dystrophin-deficient mdx mice intramuscularly. Here, we further explore the systemic potential and feasibility of PNA AOs in mediating exon skipping in mdx mice as a comprehensive systemic evaluation remains lacking. Systemic delivery of PNA AOs resulted in therapeutic level of dystrophin expression in body-wide peripheral muscles and improved dystrophic pathology in mdx mice without any detectable toxicity. Up to 40% of dystrophin restoration was achieved in gastrocnemius, to a less extent with other skeletal muscles, with no dystrophin in heart. Notably, comparable systemic activity was obtained between PNA AOs and phosphorodiamidate morpholino oligomer, a DMD AO chemistry in phase 3 clinical trial, under an identical dosing regimen. Overall, our data demonstrate that PNA is viable for DMD exon-skipping therapeutics with 20 mer showing the best combination of activity, solubility, and safety and further modifications to increase PNA aqueous solubility can enable longer, more effective therapeutics without the associated toxicity. PMID:26440599

  6. Disruption of the splicing enhancer sequence within exon 27 of the dystrophin gene by a nonsense mutation induces partial skipping of the exon and is responsible for Becker muscular dystrophy.

    PubMed Central

    Shiga, N; Takeshima, Y; Sakamoto, H; Inoue, K; Yokota, Y; Yokoyama, M; Matsuo, M

    1997-01-01

    The mechanism of exon skipping induced by nonsense mutations has not been well elucidated. We now report results of in vitro splicing studies which disclosed that a particular example of exon skipping is due to disruption of a splicing enhancer sequence located within the exon. A nonsense mutation (E1211X) due to a G to T transversion at the 28th nucleotide of exon 27 (G3839T) was identified in the dystrophin gene of a Japanese Becker muscular dystrophy case. Partial skipping of the exon resulted in the production of truncated dystrophin mRNA, although the consensus sequences for splicing at both ends of exon 27 were unaltered. To determine how E1211X induced exon 27 skipping, the splicing enhancer activity of purine-rich region within exon 27 was examined in an in vitro splicing system using chimeric doublesex gene pre-mRNA. The mutant sequence containing G3839T abolished splicing enhancer activity of the wild-type purine-rich sequence for the upstream intron in this chimeric pre-mRNA. An artificial polypurine oligonucleotide mimicking the purine-rich sequence of exon 27 also showed enhancer activity that was suppressed by the introduction of a T nucleotide. Furthermore, the splicing enhancer activity was more markedly inhibited when a nonsense codon was created by the inserted T residue. This is the first evidence that partial skipping of an exon harboring a nonsense mutation is due to disruption of a splicing enhancer sequence. PMID:9410897

  7. Disruption of the splicing enhancer sequence within exon 27 of the dystrophin gene by a nonsense mutation induces partial skipping of the exon and is responsible for Becker muscular dystrophy.

    PubMed

    Shiga, N; Takeshima, Y; Sakamoto, H; Inoue, K; Yokota, Y; Yokoyama, M; Matsuo, M

    1997-11-01

    The mechanism of exon skipping induced by nonsense mutations has not been well elucidated. We now report results of in vitro splicing studies which disclosed that a particular example of exon skipping is due to disruption of a splicing enhancer sequence located within the exon. A nonsense mutation (E1211X) due to a G to T transversion at the 28th nucleotide of exon 27 (G3839T) was identified in the dystrophin gene of a Japanese Becker muscular dystrophy case. Partial skipping of the exon resulted in the production of truncated dystrophin mRNA, although the consensus sequences for splicing at both ends of exon 27 were unaltered. To determine how E1211X induced exon 27 skipping, the splicing enhancer activity of purine-rich region within exon 27 was examined in an in vitro splicing system using chimeric doublesex gene pre-mRNA. The mutant sequence containing G3839T abolished splicing enhancer activity of the wild-type purine-rich sequence for the upstream intron in this chimeric pre-mRNA. An artificial polypurine oligonucleotide mimicking the purine-rich sequence of exon 27 also showed enhancer activity that was suppressed by the introduction of a T nucleotide. Furthermore, the splicing enhancer activity was more markedly inhibited when a nonsense codon was created by the inserted T residue. This is the first evidence that partial skipping of an exon harboring a nonsense mutation is due to disruption of a splicing enhancer sequence. PMID:9410897

  8. A novel point mutation (G-1 to T) in a 5' splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker muscular dystrophy.

    PubMed Central

    Hagiwara, Y.; Nishio, H.; Kitoh, Y.; Takeshima, Y.; Narita, N.; Wada, H.; Yokoyama, M.; Nakamura, H.; Matsuo, M.

    1994-01-01

    The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. We now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5' splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5' splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G-1-to-T mutation at the 5' splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection. Images Figure 2 Figure 5 PMID:8279470

  9. A novel point mutation (G-1 to T) in a 5' splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker muscular dystrophy.

    PubMed

    Hagiwara, Y; Nishio, H; Kitoh, Y; Takeshima, Y; Narita, N; Wada, H; Yokoyama, M; Nakamura, H; Matsuo, M

    1994-01-01

    The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. We now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5' splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5' splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G-1-to-T mutation at the 5' splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection. PMID:8279470

  10. A novel point mutation (G[sup [minus]1] to T) in a 5[prime] splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker Muscular Dystrophy

    SciTech Connect

    Hagiwara, Yoko; Nishio, Hisahide; Kitoh, Yoshihiko; Takeshima, Yasuhiro; Narita, Naoko; Wada, Hiroko; Yokoyama, Mitsuhiro; Nakamura, Hajime; Matsuo, Masafumi )

    1994-01-01

    The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. The authors now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5[prime] splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5[prime] splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G[sup [minus]1]-to-T mutation at the 5[prime] splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection. 48 refs., 5 figs.

  11. Mice Lacking Dystrophin or α Sarcoglycan Spontaneously Develop Embryonal Rhabdomyosarcoma with Cancer-Associated p53 Mutations and Alternatively Spliced or Mutant Mdm2 Transcripts

    PubMed Central

    Fernandez, Karen; Serinagaoglu, Yelda; Hammond, Sue; Martin, Laura T.; Martin, Paul T.

    2010-01-01

    Altered expression of proteins in the dystrophin-associated glycoprotein complex results in muscular dystrophy and has more recently been implicated in a number of forms of cancer. Here we show that loss of either of two members of this complex, dystrophin in mdx mice or α sarcoglycan in Sgca−/− mice, results in the spontaneous development of muscle-derived embryonal rhabdomyosarcoma (RMS) after 1 year of age. Many mdx and Sgca−/− tumors showed increased expression of insulin-like growth factor 2, retinoblastoma protein, and phosphorylated Akt and decreased expression of phosphatase and tensin homolog gene, much as is found in a human RMS. Further, all mdx and Sgca−/− RMS analyzed had increased expression of p53 and murine double minute (mdm)2 protein and contained missense p53 mutations previously identified in human cancers. The mdx RMS also contained missense mutations in Mdm2 or alternatively spliced Mdm2 transcripts that lacked an exon encoding a portion of the p53-binding domain. No Pax3:Fkhr or Pax7:Fkhr translocation mRNA products were evident in any tumor. Expression of natively glycosylated α dystroglycan and α sarcoglycan was reduced in mdx RMS, whereas dystrophin expression was absent in almost all human RMS, both for embryonal and alveolar RMS subtypes. These studies show that absence of members of the dystrophin-associated glycoprotein complex constitutes a permissive environment for spontaneous development of embryonal RMS associated with mutation of p53 and mutation or altered splicing of Mdm2. PMID:20019182

  12. Molecular aspects of DNA splicing system

    NASA Astrophysics Data System (ADS)

    Yusof, Yuhani; Lim, Wen Li; Goode, T. Elizabeth; Sarmin, Nor Haniza; Heng, Fong Wan; Wahab, Mohd Firdaus Abd

    2015-05-01

    The pioneer model of deoxyribonucleic acid (DNA) splicing system in a framework of Formal Language Theory was introduced by Head that led to the existence of other models of splicing system, namely Paun, Pixton and Yusof-Goode. These entire models are inspired by the molecular biological process of DNA splicing. Hence, this paper focuses on the translucent DNA splicing process, particularly on the generated language. Starting with some preliminaries in a limit graph, this paper also provides the experimental design with the predicted and actual result.

  13. Correlating In Vitro Splice Switching Activity With Systemic In Vivo Delivery Using Novel ZEN-modified Oligonucleotides

    PubMed Central

    Hammond, Suzan M; McClorey, Graham; Nordin, Joel Z; Godfrey, Caroline; Stenler, Sofia; Lennox, Kim A; Smith, CI Edvard; Jacobi, Ashley M; Varela, Miguel A; Lee, Yi; Behlke, Mark A; Wood, Matthew J A; Andaloussi, Samir E L

    2014-01-01

    Splice switching oligonucleotides (SSOs) induce alternative splicing of pre-mRNA and typically employ chemical modifications to increase nuclease resistance and binding affinity to target pre-mRNA. Here we describe a new SSO non-base modifier (a naphthyl-azo group, “ZEN™”) to direct exon exclusion in mutant dystrophin pre-mRNA to generate functional dystrophin protein. The ZEN modifier is placed near the ends of a 2′-O-methyl (2′OMe) oligonucleotide, increasing melting temperature and potency over unmodified 2′OMe oligonucleotides. In cultured H2K cells, a ZEN-modified 2′OMe phosphorothioate (PS) oligonucleotide delivered by lipid transfection greatly enhanced dystrophin exon skipping over the same 2′OMePS SSO lacking ZEN. However, when tested using free gymnotic uptake in vitro and following systemic delivery in vivo in dystrophin deficient mdx mice, the same ZEN-modified SSO failed to enhance potency. Importantly, we show for the first time that in vivo activity of anionic SSOs is modelled in vitro only when using gymnotic delivery. ZEN is thus a novel modifier that enhances activity of SSOs in vitro but will require improved delivery methods before its in vivo clinical potential can be realized. PMID:25423116

  14. The Characterizations of Different Splicing Systems

    NASA Astrophysics Data System (ADS)

    Karimi, Fariba; Sarmin, Nor Haniza; Heng, Fong Wan

    The concept of splicing system was first introduced by Head in 1987 to model the biological process of DNA recombination mathematically. This model was made on the basis of formal language theory which is a branch of applied discrete mathematics and theoretical computer science. In fact, splicing system treats DNA molecule and the recombinant behavior by restriction enzymes and ligases in the form of words and splicing rules respectively. The notion of splicing systems was taken into account from different points of view by many mathematicians. Several modified definitions have been introduced by many researchers. In this paper, some properties of different kinds of splicing systems are presented and their relationships are investigated. Furthermore, these results are illustrated by some examples.

  15. Dystrophin quantification

    PubMed Central

    Anthony, Karen; Arechavala-Gomeza, Virginia; Taylor, Laura E.; Vulin, Adeline; Kaminoh, Yuuki; Torelli, Silvia; Feng, Lucy; Janghra, Narinder; Bonne, Gisèle; Beuvin, Maud; Barresi, Rita; Henderson, Matt; Laval, Steven; Lourbakos, Afrodite; Campion, Giles; Straub, Volker; Voit, Thomas; Sewry, Caroline A.; Morgan, Jennifer E.; Flanigan, Kevin M.

    2014-01-01

    Objective: We formed a multi-institution collaboration in order to compare dystrophin quantification methods, reach a consensus on the most reliable method, and report its biological significance in the context of clinical trials. Methods: Five laboratories with expertise in dystrophin quantification performed a data-driven comparative analysis of a single reference set of normal and dystrophinopathy muscle biopsies using quantitative immunohistochemistry and Western blotting. We developed standardized protocols and assessed inter- and intralaboratory variability over a wide range of dystrophin expression levels. Results: Results from the different laboratories were highly concordant with minimal inter- and intralaboratory variability, particularly with quantitative immunohistochemistry. There was a good level of agreement between data generated by immunohistochemistry and Western blotting, although immunohistochemistry was more sensitive. Furthermore, mean dystrophin levels determined by alternative quantitative immunohistochemistry methods were highly comparable. Conclusions: Considering the biological function of dystrophin at the sarcolemma, our data indicate that the combined use of quantitative immunohistochemistry and Western blotting are reliable biochemical outcome measures for Duchenne muscular dystrophy clinical trials, and that standardized protocols can be comparable between competent laboratories. The methodology validated in our study will facilitate the development of experimental therapies focused on dystrophin production and their regulatory approval. PMID:25355828

  16. Systemic administration of micro-dystrophin restores cardiac geometry and prevents dobutamine-induced cardiac pump failure.

    PubMed

    Townsend, DeWayne; Blankinship, Michael J; Allen, James M; Gregorevic, Paul; Chamberlain, Jeffrey S; Metzger, Joseph M

    2007-06-01

    Duchenne muscular dystrophy (DMD) is a fatal disease of striated muscle deterioration resulting from the loss of the cytoskeletal protein dystrophin. Most patients develop significant cardiomyopathy, with heart failure being the second leading cause of death in DMD. Compared with the extensive studies on skeletal muscle defects and potential therapy in DMD, very little attention has been directed at the increasing incidence of heart failure in DMD. Here we show that a single systemic injection of recombinant adeno-associated virus (rAAV2/6) harboring micro-dystrophin leads to extensive cardiac transduction, with micro-dystrophin correctly localized at the periphery of the cardiac myocytes and functionally associated with the sarcolemmal membrane. Significantly, micro-dystrophin gene transfer corrected the baseline end-diastolic volume defect in the mdx mouse heart and prevented cardiac pump failure induced by dobutamine stress testing in vivo, although several parameters of systolic function were not corrected. These results demonstrate that systemic gene delivery of micro-dystrophin can restore ventricular distensibility and protect the mdx myocardium from pump dysfunction during adrenergic stimulation in vivo. PMID:17440445

  17. Sustained Dystrophin Expression Induced by Peptide-conjugated Morpholino Oligomers in the Muscles of mdx Mice

    PubMed Central

    Jearawiriyapaisarn, Natee; Moulton, Hong M; Buckley, Brian; Roberts, Jennifer; Sazani, Peter; Fucharoen, Suthat; Iversen, Patrick L; Kole, Ryszard

    2009-01-01

    Cell-penetrating peptides (CPPs), containing arginine (R), 6-aminohexanoic acid (X), and/or β-alanine (B) conjugated to phosphorodiamidate morpholino oligomers (PMOs), enhance their delivery in cell culture. In this study, the potency, functional biodistribution, and toxicity of these conjugates were evaluated in vivo, in EGFP-654 transgenic mice that ubiquitously express the aberrantly spliced EGFP-654 pre-mRNA reporter. Correct splicing and enhanced green fluorescence protein (EGFP) upregulation serve as a positive readout for peptide-PMO (PPMO) entry into cells and access to EGFP-654 pre-mRNA in the nucleus. Intraperitoneal injections of a series of PPMOs, A-N (12 mg/kg), administered once a day for four successive days resulted in splicing correction in numerous tissues. PPMO-B was highly potent in the heart, diaphragm, and quadriceps, which are key muscles in the treatment of Duchenne muscular dystrophy. We therefore investigated PPMO M23D-B, designed to force skipping of stop-codon containing dystrophin exon 23, in an mdx mouse model of the disease. Systemic delivery of M23D-B yielded persistent exon 23 skipping, yielding high and sustained dystrophin protein expression in body-wide muscles, including cardiac muscle, without detectable toxicity. The rescued dystrophin reduced serum creatinine kinase to near-wild-type levels, indicating improvement in muscle integrity. This is the first report of oligonucleotide-mediated exon skipping and dystrophin protein induction in the heart of treated animals. PMID:18545222

  18. Some characteristics of probabilistic one-sided splicing systems

    NASA Astrophysics Data System (ADS)

    Selvarajoo, Mathuri; Fong, Wan Heng; Sarmin, Nor Haniza; Turaev, Sherzod

    2013-04-01

    A theoretical model for DNA computing using the recombination behavior of DNA molecules known as asplicing system has been introduced in 1987. Splicing systems are based on the splicing operation which, informally, cuts two strings at the specific places and attaches the prefix of the first string to the suffix of the second string and the prefix of the second string to the suffix of the first string yielding the new strings. It is known that splicing systems with finite sets of axioms and splicing rules only generate regular languages. Hence, different types of restrictions for splicing systems have been considered to increase the computational power of the languages generated. Recently, probabilistic splicing systems have been introduced where the probabilities are initially associated with the axioms, and the probabilities of the generated strings are computed from the probabilities of the initial strings. In this paper, some properties of probabilistic one-sided splicing systems, which are special types of probabilistic splicing systems, are investigated. We prove that probabilistic one-sided splicing systems can also increase the computational power of the languages generated.

  19. A quantitative ELISA for dystrophin.

    PubMed

    Morris, G E; Ellis, J M; Nguyen, T M

    1993-05-01

    A novel approach to the quantitation of the muscular dystrophy protein, dystrophin, in muscle extracts is described. The two-site ELISA uses two monoclonal antibodies against dystrophin epitopes which lie close together in the rod domain of the dystrophin molecule in order to minimize the effects of dystrophin degradation. Dystrophin is assayed in its native form by extracting with non-ionic detergents and avoiding the use of SDS. PMID:8486926

  20. Ocular and neurodevelopmental features of Duchenne muscular dystrophy: a signature of dystrophin function in the central nervous system.

    PubMed

    Ricotti, Valeria; Jägle, Herbert; Theodorou, Maria; Moore, Anthony T; Muntoni, Francesco; Thompson, Dorothy A

    2016-04-01

    Multiple isoforms of dystrophin (Dp427, Dp260, Dp140, Dp71) are expressed differentially in the central nervous system (CNS) including the retinal layers. Disruption of these protein products is responsible for cognitive dysfunction, electroretinogram (ERG) abnormalities and behavioural disorders in Duchenne muscular dystrophy (DMD). We studied the ocular characteristics and neuropsychiatric profile of 16 DMD boys. The ISCEV standard, full-field flash ERGs were assessed. Intellectual ability and behavioural disturbances were measured. All genotypes were associated with mildly abnormal photopic ERG a:b-wave amplitude ratios. In addition, we identified the following genotype/phenotype correlations: boys with mutations upstream of exon 30 (ie, isolated Dp427 altered expression) showed normal scotopic a:b ratios, abnormal photopic oscillatory potential OP2 and normal scotopic OP2. Conversely, all boys with DMD mutations downstream of exon 30 showed profoundly 'negative' scotopic ERGs (a:b ratios >1). In these patients, the involvement of Dp260 isoform resulted in the absence of slow rod pathway signalling in15 Hz scotopic flicker ERGs. These boys had abnormal scotopic OP2 and normal photopic OP2. Finally, children with mutations also affecting Dp71 were associated with more pronounced electronegative ERGs. When correlating ERGs to neurodevelopmental outcome, we found a positive correlation between negative scotopic ERGs and neurodevelopmental disturbances, and the most severe findings were in boys with Dp71 disruption. These findings suggest a strong association between DMD mutations affecting different DMD isoforms with characteristically abnormal scotopic ERGs and severe neurodevelopmental problems. The role of the ERG as a potential biomarker for dystrophin function in the CNS and response to novel genetic therapies warrants further exploration. PMID:26081639

  1. Effects of irradiating adult mdx mice before full-length dystrophin cDNA transfer on host anti-dystrophin immunity.

    PubMed

    Eghtesad, S; Zheng, H; Nakai, H; Epperly, M W; Clemens, P R

    2010-09-01

    Duchenne muscular dystrophy is a fatal, genetic disorder in which dystrophin-deficient muscle progressively degenerates, for which dystrophin gene transfer could provide effective treatment. The host immune response to dystrophin, however, is an obstacle to therapeutic gene expression. Understanding the dystrophin-induced host immune response will facilitate the discovery of strategies to prolong expression of recombinant dystrophin in dystrophic muscle. Using whole-body irradiation of the dystrophic mdx mouse before gene transfer, we temporally removed the immune system; a 600 rad dose removed peripheral immune cells, which were restored by self-reconstitution, and a 900 rad dose removed central and peripheral immune cells, which were restored by adoptive transfer of bone marrow from a syngeneic, dystrophin-normal donor. The anti-dystrophin humoral response was delayed and dystrophin expression was partially preserved in irradiated, vector-treated mice. Nonirradiated, vector-treated control mice lost muscle dystrophin expression completely, had an earlier anti-dystrophin humoral response and demonstrated muscle fibers focally surrounded with T cells. We conclude that dystrophin gene transfer induced anti-dystrophin humoral immunity and cell-mediated responses that were significantly diminished and delayed by temporal removal of the host central or peripheral immune cells. Furthermore, manipulation of central immunity altered the pattern of regulatory T cells in muscle. PMID:20827278

  2. Long-Term Efficacy of Systemic Multiexon Skipping Targeting Dystrophin Exons 45–55 With a Cocktail of Vivo-Morpholinos in Mdx52 Mice

    PubMed Central

    Echigoya, Yusuke; Aoki, Yoshitsugu; Miskew, Bailey; Panesar, Dharminder; Touznik, Aleksander; Nagata, Tetsuya; Tanihata, Jun; Nakamura, Akinori; Nagaraju, Kanneboyina; Yokota, Toshifumi

    2015-01-01

    Antisense-mediated exon skipping, which can restore the reading frame, is a most promising therapeutic approach for Duchenne muscular dystrophy. Remaining challenges include the limited applicability to patients and unclear function of truncated dystrophin proteins. Multiexon skipping targeting exons 45–55 at the mutation hotspot of the dystrophin gene could overcome both of these challenges. Previously, we described the feasibility of exons 45–55 skipping with a cocktail of Vivo-Morpholinos in vivo; however, the long-term efficacy and safety of Vivo-Morpholinos remains to be determined. In this study, we examined the efficacy and toxicity of exons 45–55 skipping by intravenous injections of 6 mg/kg 10-Vivo-Morpholino cocktail (0.6 mg/kg each vPMO) every 2 weeks for 18 weeks to dystrophic exon-52 knockout (mdx52) mice. Systemic skipping of the entire exons 45–55 region was induced, and the Western blot analysis exhibited the restoration of 5–27% of normal levels of dystrophin protein in skeletal muscles, accompanied by improvements in histopathology and muscle strength. No obvious immune response and renal and hepatic toxicity were detected at the end-point of the treatment. We demonstrate our new regimen with the 10-Vivo-Morpholino cocktail is effective and safe for long-term repeated systemic administration in the dystrophic mouse model. PMID:25647512

  3. Long-term efficacy of systemic multiexon skipping targeting dystrophin exons 45-55 with a cocktail of vivo-morpholinos in mdx52 mice.

    PubMed

    Echigoya, Yusuke; Aoki, Yoshitsugu; Miskew, Bailey; Panesar, Dharminder; Touznik, Aleksander; Nagata, Tetsuya; Tanihata, Jun; Nakamura, Akinori; Nagaraju, Kanneboyina; Yokota, Toshifumi

    2015-01-01

    Antisense-mediated exon skipping, which can restore the reading frame, is a most promising therapeutic approach for Duchenne muscular dystrophy. Remaining challenges include the limited applicability to patients and unclear function of truncated dystrophin proteins. Multiexon skipping targeting exons 45-55 at the mutation hotspot of the dystrophin gene could overcome both of these challenges. Previously, we described the feasibility of exons 45-55 skipping with a cocktail of Vivo-Morpholinos in vivo; however, the long-term efficacy and safety of Vivo-Morpholinos remains to be determined. In this study, we examined the efficacy and toxicity of exons 45-55 skipping by intravenous injections of 6 mg/kg 10-Vivo-Morpholino cocktail (0.6 mg/kg each vPMO) every 2 weeks for 18 weeks to dystrophic exon-52 knockout (mdx52) mice. Systemic skipping of the entire exons 45-55 region was induced, and the Western blot analysis exhibited the restoration of 5-27% of normal levels of dystrophin protein in skeletal muscles, accompanied by improvements in histopathology and muscle strength. No obvious immune response and renal and hepatic toxicity were detected at the end-point of the treatment. We demonstrate our new regimen with the 10-Vivo-Morpholino cocktail is effective and safe for long-term repeated systemic administration in the dystrophic mouse model. PMID:25647512

  4. Exon skipping and translation in patients with frameshift deletions in the dystrophin gene

    SciTech Connect

    Sherratt, T.G.; Dubowitz, V.; Sewry, C.A.; Strong, P.N. ); Vulliamy, T. )

    1993-11-01

    Although many Duchenne muscular dystrophy patients have a deletion in the dystrophin gene which disrupts the translational reading frame, they express dystrophin in a small proportion of skeletal muscle fibers ([open quotes]revertant fibers[close quotes]). Antibody studies have shown, indirectly, that dystrophin synthesis in revertant fibers is facilitated by a frame-restoring mechanism; in the present study, the feasibility of mRNA splicing was investigated. Dystrophin transcripts were analyzed in skeletal muscle from individuals possessing revertant fibers and a frameshift deletion in the dystrophin gene. In each case a minor in-frame transcript was detected, in which exons adjacent to those deleted from the genome had been skipped. There appeared to be some correlation between the levels of in-frame transcripts and the predicted translation products. Low levels of alternatively spliced transcripts were also present in normal muscle. The results provide further evidence of exon skipping in the dystrophin gene and indicate that this may be involved in the synthesis of dystrophin by revertant fibers. 44 refs., 12 figs.

  5. How much dystrophin is enough: the physiological consequences of different levels of dystrophin in the mdx mouse.

    PubMed

    Godfrey, Caroline; Muses, Sofia; McClorey, Graham; Wells, Kim E; Coursindel, Thibault; Terry, Rebecca L; Betts, Corinne; Hammond, Suzan; O'Donovan, Liz; Hildyard, John; El Andaloussi, Samir; Gait, Michael J; Wood, Matthew J; Wells, Dominic J

    2015-08-01

    Splice modulation therapy has shown great clinical promise in Duchenne muscular dystrophy, resulting in the production of dystrophin protein. Despite this, the relationship between restoring dystrophin to established dystrophic muscle and its ability to induce clinically relevant changes in muscle function is poorly understood. In order to robustly evaluate functional improvement, we used in situ protocols in the mdx mouse to measure muscle strength and resistance to eccentric contraction-induced damage. Here, we modelled the treatment of muscle with pre-existing dystrophic pathology using antisense oligonucleotides conjugated to a cell-penetrating peptide. We reveal that 15% homogeneous dystrophin expression is sufficient to protect against eccentric contraction-induced injury. In addition, we demonstrate a >40% increase in specific isometric force following repeated administrations. Strikingly, we show that changes in muscle strength are proportional to dystrophin expression levels. These data define the dystrophin restoration levels required to slow down or prevent disease progression and improve overall muscle function once a dystrophic environment has been established in the mdx mouse model. PMID:25935000

  6. Dystrophin in frameshift deletion patients with Becker Muscular Dystrophy

    SciTech Connect

    Gangopadhyay, S.B.; Ray, P.N.; Worton, R.G.; Sherratt, T.G.; Heckmatt, J.Z.; Dubowitz, V.; Strong, P.N.; Miller, G. ); Shokeir, M. )

    1992-09-01

    In a previous study the authors identified 14 cases with Duchenne muscular dystrophy (DMD) or its milder variant, Becker muscular dystrophy (BMD), with a deletion of exons 3-7, a deletion that would be expected to shift the translational reading frame of the mRNA and give a severe phenotype. They have examined dystrophin and its mRNA from muscle biopsies of seven cases with either mild or intermediate phenotypes. In all cases they detected slightly lower-molecular-weight dystrophin in 12%-15% abundance relative to the normal. By sequencing amplified mRNA they have found that exon 2 is spliced to exon 8, a splice that produces a frameshifted mRNA, and have found no evidence for alternate splicing that might be involved in restoration of dystrophin mRNA reading frame in the patients with a mild phenotype. Other transcriptional and posttranscriptional mechanisms such as cryptic promoter, ribosomal frameshifting, and reinitiation are suggested that might play some role in restoring the reading frame. 34 refs., 5 figs. 1 tab.

  7. Autologous skeletal muscle derived cells expressing a novel functional dystrophin provide a potential therapy for Duchenne Muscular Dystrophy

    PubMed Central

    Meng, Jinhong; Counsell, John R.; Reza, Mojgan; Laval, Steven H.; Danos, Olivier; Thrasher, Adrian; Lochmüller, Hanns; Muntoni, Francesco; Morgan, Jennifer E.

    2016-01-01

    Autologous stem cells that have been genetically modified to express dystrophin are a possible means of treating Duchenne Muscular Dystrophy (DMD). To maximize the therapeutic effect, dystrophin construct needs to contain as many functional motifs as possible, within the packaging capacity of the viral vector. Existing dystrophin constructs used for transduction of muscle stem cells do not contain the nNOS binding site, an important functional motif within the dystrophin gene. In this proof-of-concept study, using stem cells derived from skeletal muscle of a DMD patient (mdcs) transplanted into an immunodeficient mouse model of DMD, we report that two novel dystrophin constructs, C1 (ΔR3-R13) and C2 (ΔH2-R23), can be lentivirally transduced into mdcs and produce dystrophin. These dystrophin proteins were functional in vivo, as members of the dystrophin glycoprotein complex were restored in muscle fibres containing donor-derived dystrophin. In muscle fibres derived from cells that had been transduced with construct C1, the largest dystrophin construct packaged into a lentiviral system, nNOS was restored. The combination of autologous stem cells and a lentivirus expressing a novel dystrophin construct which optimally restores proteins of the dystrophin glycoprotein complex may have therapeutic application for all DMD patients, regardless of their dystrophin mutation. PMID:26813695

  8. Autologous skeletal muscle derived cells expressing a novel functional dystrophin provide a potential therapy for Duchenne Muscular Dystrophy.

    PubMed

    Meng, Jinhong; Counsell, John R; Reza, Mojgan; Laval, Steven H; Danos, Olivier; Thrasher, Adrian; Lochmüller, Hanns; Muntoni, Francesco; Morgan, Jennifer E

    2016-01-01

    Autologous stem cells that have been genetically modified to express dystrophin are a possible means of treating Duchenne Muscular Dystrophy (DMD). To maximize the therapeutic effect, dystrophin construct needs to contain as many functional motifs as possible, within the packaging capacity of the viral vector. Existing dystrophin constructs used for transduction of muscle stem cells do not contain the nNOS binding site, an important functional motif within the dystrophin gene. In this proof-of-concept study, using stem cells derived from skeletal muscle of a DMD patient (mdcs) transplanted into an immunodeficient mouse model of DMD, we report that two novel dystrophin constructs, C1 (ΔR3-R13) and C2 (ΔH2-R23), can be lentivirally transduced into mdcs and produce dystrophin. These dystrophin proteins were functional in vivo, as members of the dystrophin glycoprotein complex were restored in muscle fibres containing donor-derived dystrophin. In muscle fibres derived from cells that had been transduced with construct C1, the largest dystrophin construct packaged into a lentiviral system, nNOS was restored. The combination of autologous stem cells and a lentivirus expressing a novel dystrophin construct which optimally restores proteins of the dystrophin glycoprotein complex may have therapeutic application for all DMD patients, regardless of their dystrophin mutation. PMID:26813695

  9. Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study

    PubMed Central

    Kinali, Maria; Arechavala-Gomeza, Virginia; Feng, Lucy; Cirak, Sebahattin; Hunt, David; Adkin, Carl; Guglieri, Michela; Ashton, Emma; Abbs, Stephen; Nihoyannopoulos, Petros; Garralda, Maria Elena; Rutherford, Mary; Mcculley, Caroline; Popplewell, Linda; Graham, Ian R; Dickson, George; Wood, Matthew JA; Wells, Dominic J; Wilton, Steve D; Kole, Ryszard; Straub, Volker; Bushby, Kate; Sewry, Caroline; Morgan, Jennifer E; Muntoni, Francesco

    2009-01-01

    Summary Background Mutations that disrupt the open reading frame and prevent full translation of DMD, the gene that encodes dystrophin, underlie the fatal X-linked disease Duchenne muscular dystrophy. Oligonucleotides targeted to splicing elements (splice switching oligonucleotides) in DMD pre-mRNA can lead to exon skipping, restoration of the open reading frame, and the production of functional dystrophin in vitro and in vivo, which could benefit patients with this disorder. Methods We did a single-blind, placebo-controlled, dose-escalation study in patients with DMD recruited nationally, to assess the safety and biochemical efficacy of an intramuscular morpholino splice-switching oligonucleotide (AVI-4658) that skips exon 51 in dystrophin mRNA. Seven patients with Duchenne muscular dystrophy with deletions in the open reading frame of DMD that are responsive to exon 51 skipping were selected on the basis of the preservation of their extensor digitorum brevis (EDB) muscle seen on MRI and the response of cultured fibroblasts from a skin biopsy to AVI-4658. AVI-4658 was injected into the EDB muscle; the contralateral muscle received saline. Muscles were biopsied between 3 and 4 weeks after injection. The primary endpoint was the safety of AVI-4658 and the secondary endpoint was its biochemical efficacy. This trial is registered, number NCT00159250. Findings Two patients received 0·09 mg AVI-4658 in 900 μL (0·9%) saline and five patients received 0·9 mg AVI-4658 in 900 μL saline. No adverse events related to AVI-4658 administration were reported. Intramuscular injection of the higher-dose of AVI-4658 resulted in increased dystrophin expression in all treated EDB muscles, although the results of the immunostaining of EDB-treated muscle for dystrophin were not uniform. In the areas of the immunostained sections that were adjacent to the needle track through which AVI-4658 was given, 44–79% of myofibres had increased expression of dystrophin. In randomly chosen

  10. Alterations of dystrophin-associated glycoproteins in the heart lacking dystrophin or dystrophin and utrophin.

    PubMed

    Sharpe, Katharine M; Premsukh, Monica D; Townsend, DeWayne

    2013-12-01

    Heart disease is a leading cause of death in patients with Duchenne muscular dystrophy (DMD). Patients with DMD lack the protein dystrophin, which is widely expressed in striated muscle. In skeletal muscle, the loss of dystrophin results in dramatically decreased expression of the dystrophin associated glycoprotein complex (DGC). Interestingly, in the heart the DGC is normally expressed without dystrophin; this has been attributed to presence of the dystrophin homologue utrophin. We demonstrate here that neither utrophin nor dystrophin are required for the expression of the cardiac DGC. However, alpha-dystroglycan (α-DG), a major component of the DGC, is differentially glycosylated in dystrophin-(mdx) and dystrophin-/utrophin-(dko) deficient mouse hearts. In both models the altered α-DG retains laminin binding activity, but has an altered localization at the sarcolemma. In hearts lacking both dystrophin and utrophin, the alterations in α-DG glycosylation are even more dramatic with changes in gel migration equivalent to 24 ± 3 kDa. These data show that the absence of dystrophin and utrophin alters the processing of α-DG; however it is not clear if these alterations are a consequence of the loss of a direct interaction with dystrophin/utrophin or results from an indirect response to the presence of severe pathology. Recently there have been great advances in our understanding of the glycosylation of α-DG regarding its role as a laminin receptor. Here we present data that alterations in glycosylation occur in the hearts of animal models of DMD, but these changes do not affect laminin binding. The physiological consequences of these alterations remain unknown, but may have significant implications for the development of therapies for DMD. PMID:24096570

  11. A family with a dystrophin gene mutation specifically affecting dystrophin expression in the heart

    SciTech Connect

    Muntoni, F.; Davies, K.; Dubowitz, V.

    1994-09-01

    We recently described a family with X-linked dilated cardiomyopathy where a large deletion in the muscle promoter region of the dystrophin gene was associated with a severe dilated cardiomyopathy in absence of clinical skeletal muscle involvement. The deletion removed the entire muscle promoter region, the first muscle exon and part of intron 1. The brain and Purkinje cell promoters were not affected by the deletion. Despite the lack of both the muscle promoter and the first muscle exon, dystrophin was detected immunocytochemically in relative high levels in the skeletal muscle of the affected males. We have now found that both the brain and Purkinje cell promoters were transcribed at high levels in the skeletal muscle of these individuals. This phenomenon, that does not occur in normal skeletal muscle, indicates that these two isoforms, physiologically expressed mainly in the central nervous system, can be transcribed and be functionally active in skeletal muscle under specific circumstances. Contrary to what is observed in skeletal muscle, dystrophin was not detected in the heart of one affected male using immunocytochemistry and an entire panel of anti-dystrophin antibodies. This was most likely the cause for the pronounced cardiac fibrosis observed and eventually responsible for the severe cardiac involvement invariably seen in seven affected males. In conclusion, the mutation of the muscle promoter, first muscle exon and part of intron 1 specifically affected expression of dystrophin in the heart. We believe that this deletion removes sequences involved in regulation of dystrophin expression in the heart and are at the moment characterizing other families with X-linked cardiomyopathy secondary to a dystrophinopathy.

  12. Correction of Dystrophin Expression in Cells From Duchenne Muscular Dystrophy Patients Through Genomic Excision of Exon 51 by Zinc Finger Nucleases

    PubMed Central

    Ousterout, David G; Kabadi, Ami M; Thakore, Pratiksha I; Perez-Pinera, Pablo; Brown, Matthew T; Majoros, William H; Reddy, Timothy E; Gersbach, Charles A

    2015-01-01

    Duchenne muscular dystrophy (DMD) is caused by genetic mutations that result in the absence of dystrophin protein expression. Oligonucleotide-induced exon skipping can restore the dystrophin reading frame and protein production. However, this requires continuous drug administration and may not generate complete skipping of the targeted exon. In this study, we apply genome editing with zinc finger nucleases (ZFNs) to permanently remove essential splicing sequences in exon 51 of the dystrophin gene and thereby exclude exon 51 from the resulting dystrophin transcript. This approach can restore the dystrophin reading frame in ~13% of DMD patient mutations. Transfection of two ZFNs targeted to sites flanking the exon 51 splice acceptor into DMD patient myoblasts led to deletion of this genomic sequence. A clonal population was isolated with this deletion and following differentiation we confirmed loss of exon 51 from the dystrophin mRNA transcript and restoration of dystrophin protein expression. Furthermore, transplantation of corrected cells into immunodeficient mice resulted in human dystrophin expression localized to the sarcolemmal membrane. Finally, we quantified ZFN toxicity in human cells and mutagenesis at predicted off-target sites. This study demonstrates a powerful method to restore the dystrophin reading frame and protein expression by permanently deleting exons. PMID:25492562

  13. Alterations of dystrophin associated glycoproteins in the heart lacking dystrophin or dystrophin and utrophin

    PubMed Central

    Sharpe, Katharine M.; Premsukh, Monica D.; Townsend, DeWayne

    2013-01-01

    Heart disease is a leading cause of death in patients with Duchenne muscular dystrophy (DMD). Patients with DMD lack the protein dystrophin, which is widely expressed in striated muscle. In skeletal muscle, the loss of dystrophin results in dramatically decreased expression of the dystrophin associated glycoprotein complex (DGC). Interestingly, in the heart the DGC is normally expressed without dystrophin; this has been attributed to presence of the dystrophin homologue utrophin. We demonstrate here that neither utrophin nor dystrophin are required for the expression of the cardiac DGC. However, alpha-dystroglycan (α-DG), a major component of the DGC, is differentially glycosylated in dystrophin-(mdx) and dystrophin−/utrophin− (dko) deficient mouse hearts. In both models the altered α-DG retains laminin binding activity, but has an altered localization at the sarcolemma. In hearts lacking both dystrophin and utrophin, the alterations in α-DG glycosylation are even more dramatic with changes in gel migration equivalent to 24 ± 3 kDa. These data show that the absence of dystrophin and utrophin alters the processing of α-DG; however it is not clear if these alterations are a consequence of the loss of a direct interaction with dystrophin/utrophin, or results from an indirect response to the presence of severe pathology. Recently there have been great advances in our understanding of the glycosylation of α-DG regarding its role as a laminin receptor. Here we present data that alterations in glycosylation occurs in the hearts of animal models of DMD, but these changes do not affect laminin binding. The physiological consequences of these alterations remain to be determined, but may have significant implications for the development of therapies for DMD. PMID:24096570

  14. Dystrophin Distribution and Expression in Human and Experimental Temporal Lobe Epilepsy

    PubMed Central

    Hendriksen, Ruben G. F.; Schipper, Sandra; Hoogland, Govert; Schijns, Olaf E. M. G.; Dings, Jim T. A.; Aalbers, Marlien W.; Vles, Johan S. H.

    2016-01-01

    Objective: Dystrophin is part of a protein complex that connects the cytoskeleton to the extracellular matrix. In addition to its role in muscle tissue, it functions as an anchoring protein within the central nervous system such as in hippocampus and cerebellum. Its presence in the latter regions is illustrated by the cognitive problems seen in Duchenne Muscular Dystrophy (DMD). Since epilepsy is also supposed to constitute a comorbidity of DMD, it is hypothesized that dystrophin plays a role in neuronal excitability. Here, we aimed to study brain dystrophin distribution and expression in both, human and experimental temporal lobe epilepsy (TLE). Method: Regional and cellular dystrophin distribution was evaluated in both human and rat hippocampi and in rat cerebellar tissue by immunofluorescent colocalization with neuronal (NeuN and calbindin) and glial (GFAP) markers. In addition, hippocampal dystrophin levels were estimated by Western blot analysis in biopsies from TLE patients, post-mortem controls, amygdala kindled (AK)-, and control rats. Results: Dystrophin was expressed in all hippocampal pyramidal subfields and in the molecular-, Purkinje-, and granular cell layer of the cerebellum. In these regions it colocalized with GFAP, suggesting expression in astrocytes such as Bergmann glia (BG) and velate protoplasmic astrocytes. In rat hippocampus and cerebellum there were neither differences in dystrophin positive cell types, nor in the regional dystrophin distribution between AK and control animals. Quantitatively, hippocampal full-length dystrophin (Dp427) levels were about 60% higher in human TLE patients than in post-mortem controls (p < 0.05), whereas the level of the shorter Dp71 isoform did not differ. In contrast, AK animals showed similar dystrophin levels as controls. Conclusion: Dystrophin is ubiquitously expressed by astrocytes in the human and rat hippocampus and in the rat cerebellum. Hippocampal full-length dystrophin (Dp427) levels are upregulated

  15. Remote Management for Multipoint Sensing Systems Using Hetero-Core Spliced Optical Fiber Sensors

    PubMed Central

    Goh, Lee See; Anoda, Yuji; Kazuhiro, Watanabe; Shinomiya, Norihiko

    2014-01-01

    This paper describes the design and experimental verification of a multipoint sensing system with hetero-core spliced optical fiber sensors and its remote management using an internet-standard protocol. The study proposes two different types of design and conducts experiments to verify those systems' feasibility. In order to manage the sensing systems remotely, the management method uses a standard operation and maintenance protocol for internet: the Simple Network Management Protocol is proposed. The purpose of this study is to construct a multipoint sensing system remote management tool by which the system can also determine the status and the identity of fiber optic sensors. The constructed sensing systems are verified and the results have demonstrated that the first proposed system can distinguish the responses from different hetero-core spliced optical fiber sensors remotely. The second proposed system shows that data communications are performed successfully while identifying the status of hetero-core spliced optical fiber sensors remotely. PMID:24379051

  16. Restoration of dystrophin-associated proteins in skeletal muscle of mdx mice transgenic for dystrophin gene.

    PubMed

    Matsumura, K; Lee, C C; Caskey, C T; Campbell, K P

    1993-04-12

    Duchenne muscular dystrophy (DMD) patients and mdx mice are characterized by the absence of dystrophin, a membrane cytoskeletal protein. Dystrophin is associated with a large oligomeric complex of sarcolemmal glycoproteins, including dystroglycan which provides a linkage to the extracellular matrix component, laminin. The finding that all of the dystrophin-associated proteins (DAPs) are drastically reduced in DMD and mdx skeletal muscle supports the primary function of dystrophin as an anchor of the sarcolemmal glycoprotein complex to the subsarcolemmal cytoskeleton. These findings indicate that the efficacy of dystrophin gene therapy will depend not only on replacing dystrophin but also on restoring all of the DAPs in the sarcolemma. Here we have investigated the status of the DAPs in the skeletal muscle of mdx mice transgenic for the dystrophin gene. Our results demonstrate that transfer of dystrophin gene restores all of the DAPs together with dystrophin, suggesting that dystrophin gene therapy should be effective in restoring the entire dystrophin-glycoprotein complex. PMID:8462697

  17. Dystrophin-Deficient Cardiomyopathy.

    PubMed

    Kamdar, Forum; Garry, Daniel J

    2016-05-31

    Dystrophinopathies are a group of distinct neuromuscular diseases that result from mutations in the structural cytoskeletal Dystrophin gene. Dystrophinopathies include Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), X-linked dilated cardiomyopathy, as well as DMD and BMD female carriers. The primary presenting symptom in most dystrophinopathies is skeletal muscle weakness. However, cardiac muscle is also a subtype of striated muscle and is similarly affected in many of the muscular dystrophies. Cardiomyopathies associated with dystrophinopathies are an increasingly recognized manifestation of these neuromuscular disorders and contribute significantly to their morbidity and mortality. Recent studies suggest that these patient populations would benefit from cardiovascular therapies, annual cardiovascular imaging studies, and close follow-up with cardiovascular specialists. Moreover, patients with DMD and BMD who develop end-stage heart failure may benefit from the use of advanced therapies. This review focuses on the pathophysiology, cardiac involvement, and treatment of cardiomyopathy in the dystrophic patient. PMID:27230049

  18. Dual Myostatin and Dystrophin Exon Skipping by Morpholino Nucleic Acid Oligomers Conjugated to a Cell-penetrating Peptide Is a Promising Therapeutic Strategy for the Treatment of Duchenne Muscular Dystrophy

    PubMed Central

    Malerba, Alberto; Kang, Jagjeet K; McClorey, Graham; Saleh, Amer F; Popplewell, Linda; Gait, Michael J; Wood, Matthew JA; Dickson, George

    2012-01-01

    The knockdown of myostatin, a negative regulator of skeletal muscle mass may have important implications in disease conditions accompanied by muscle mass loss like cancer, HIV/AIDS, sarcopenia, muscle atrophy, and Duchenne muscular dystrophy (DMD). In DMD patients, where major muscle loss has occurred due to a lack of dystrophin, the therapeutic restoration of dystrophin expression alone in older patients may not be sufficient to restore the functionality of the muscles. We recently demonstrated that phosphorodiamidate morpholino oligomers (PMOs) can be used to re-direct myostatin splicing and promote the expression of an out-of-frame transcript so reducing the amount of the synthesized myostatin protein. Furthermore, the systemic administration of the same PMO conjugated to an octaguanidine moiety (Vivo-PMO) led to a significant increase in the mass of soleus muscle of treated mice. Here, we have further optimized the use of Vivo-PMO in normal mice and also tested the efficacy of the same PMO conjugated to an arginine-rich cell-penetrating peptide (B-PMO). Similar experiments conducted in mdx dystrophic mice showed that B-PMO targeting myostatin is able to significantly increase the tibialis anterior (TA) muscle weight and when coadministered with a B-PMO targeting the dystrophin exon 23, it does not have a detrimental interaction. This study confirms that myostatin knockdown by exon skipping is a potential therapeutic strategy to counteract muscle wasting conditions and dual myostatin and dystrophin skipping has potential as a therapy for DMD. PMID:23250360

  19. Frameshift deletions of exons 3-7 and revertant fibers in Duchenne muscular dystrophy: mechanisms of dystrophin production.

    PubMed Central

    Winnard, A V; Mendell, J R; Prior, T W; Florence, J; Burghes, A H

    1995-01-01

    Duchenne muscular dystrophy (DMD) patients with mutations that disrupt the translational reading frame produce little or no dystrophin. Two exceptions are the deletion of exons 3-7 and the occurrence of rare dystrophin-positive fibers (revertant fibers) in muscle of DMD patients. Antibodies directed against the amino-terminus and the 5' end of exon 8 did not detect dystrophin in muscle from patients who have a deletion of exons 3-7. However, in all cases, dystrophin was detected with an antibody directed against the 3' end of exon 8. The most likely method of dystrophin production in these cases is initiation at a new start codon in exon 8. We also studied two patients who have revertant fibers: one had an inherited duplication of exons 5-7, which, on immunostaining, showed two types of revertant fibers; and the second patient had a 2-bp nonsense mutation in exon 51, which creates a cryptic splice site. An in-frame mRNA that uses this splice site in exon 51 was detected. Immunostaining demonstrated the presence of the 3' end of exon 51, which is in agreement with the use of this mRNA in revertant fibers. The most likely method of dystrophin production in these fibers is a second mutation that restores the reading frame. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7825572

  20. Essential roles for the splicing regulator nSR100/SRRM4 during nervous system development

    PubMed Central

    Quesnel-Vallières, Mathieu; Irimia, Manuel

    2015-01-01

    Alternative splicing (AS) generates vast transcriptomic complexity in the vertebrate nervous system. However, the extent to which trans-acting splicing regulators and their target AS regulatory networks contribute to nervous system development is not well understood. To address these questions, we generated mice lacking the vertebrate- and neural-specific Ser/Arg repeat-related protein of 100 kDa (nSR100/SRRM4). Loss of nSR100 impairs development of the central and peripheral nervous systems in part by disrupting neurite outgrowth, cortical layering in the forebrain, and axon guidance in the corpus callosum. Accompanying these developmental defects are widespread changes in AS that primarily result in shifts to nonneural patterns for different classes of splicing events. The main component of the altered AS program comprises 3- to 27-nucleotide (nt) neural microexons, an emerging class of highly conserved AS events associated with the regulation of protein interaction networks in developing neurons and neurological disorders. Remarkably, inclusion of a 6-nt, nSR100-activated microexon in Unc13b transcripts is sufficient to rescue a neuritogenesis defect in nSR100 mutant primary neurons. These results thus reveal critical in vivo neurodevelopmental functions of nSR100 and further link these functions to a conserved program of neuronal microexon splicing. PMID:25838543

  1. HEK293 cells express dystrophin Dp71 with nucleus-specific localization of Dp71ab.

    PubMed

    Nishida, Atsushi; Yasuno, Sato; Takeuchi, Atsuko; Awano, Hiroyuki; Lee, Tomoko; Niba, Emma Tabe Eko; Fujimoto, Takahiro; Itoh, Kyoko; Takeshima, Yasuhiro; Nishio, Hisahide; Matsuo, Masafumi

    2016-09-01

    The dystrophin gene consists of 79 exons and encodes tissue-specific isoforms. Mutations in the dystrophin gene cause Duchenne muscular dystrophy, of which a substantial proportion of cases are complicated by non-progressive mental retardation. Abnormalities of Dp71, an isoform transcribed from a promoter in intron 62, are a suspected cause of mental retardation. However, the roles of Dp71 in human brain have not been fully elucidated. Here, we characterized dystrophin in human HEK293 cells with the neuronal lineage. Reverse transcription-PCR amplification of the full-length dystrophin transcript revealed the absence of fragments covering the 5' part of the dystrophin cDNA. In contrast, fragments covering exons 64-79 were present. The Dp71 promoter-specific exon G1 was shown spliced to exon 63. We demonstrated that the Dp71 transcript comprised two subisoforms: one lacking exon 78 (Dp71b) and the other lacking both exons 71 and 78 (Dp71ab). Western blotting of cell lysates using an antibody against the dystrophin C-terminal region revealed two bands, corresponding to Dp71b and Dp71ab. Immunohistochemical examination with the dystrophin antibody revealed scattered punctate signals in the cytoplasm and the nucleus. Western blotting revealed one band corresponding to Dp71b in the cytoplasm and two bands corresponding to Dp71b and Dp71ab in the nucleus, with Dp71b being predominant. These results indicated that Dp71ab is a nucleus-specific subisoform. We concluded that Dp71, comprising Dp71b and Dp71ab, was expressed exclusively in HEK293 cells and that Dp71ab was specifically localized to the nucleus. Our findings suggest that Dp71ab in the nucleus contributes to the diverse functions of HEK293 cells. PMID:27109495

  2. Metabolic profiles of dystrophin and utrophin expression in mouse models of Duchenne muscular dystrophy.

    PubMed

    Griffin, J L; Sang, E; Evens, T; Davies, K; Clarke, K

    2002-10-23

    Metabolic profiles from (1)H nuclear magnetic resonance spectroscopy have been used to describe both one and two protein systems in four mouse models related to Duchenne muscular dystrophy using the pattern recognition technique partial least squares. Robust statistical models were built for extracts and intact cardiac tissue, distinguishing mice according to expression of dystrophin. Using metabolic profiles of diaphragm, models were built describing dystrophin and utrophin, a dystrophin related protein, expression. Increased utrophin expression counteracted some of the deficits associated with dystrophic tissue. This suggests the method may be ideal for following treatment regimes such as gene therapy. PMID:12387876

  3. Splicing Regulation of Pro-Inflammatory Cytokines and Chemokines: At the Interface of the Neuroendocrine and Immune Systems.

    PubMed

    Shakola, Felitsiya; Suri, Parul; Ruggiu, Matteo

    2015-01-01

    Alternative splicing plays a key role in posttranscriptional regulation of gene expression, allowing a single gene to encode multiple protein isoforms. As such, alternative splicing amplifies the coding capacity of the genome enormously, generates protein diversity, and alters protein function. More than 90% of human genes undergo alternative splicing, and alternative splicing is especially prevalent in the nervous and immune systems, tissues where cells need to react swiftly and adapt to changes in the environment through carefully regulated mechanisms of cell differentiation, migration, targeting, and activation. Given its prevalence and complexity, this highly regulated mode of gene expression is prone to be affected by disease. In the following review, we look at how alternative splicing of signaling molecules—cytokines and their receptors—changes in different pathological conditions, from chronic inflammation to neurologic disorders, providing means of functional interaction between the immune and neuroendocrine systems. Switches in alternative splicing patterns can be very dynamic and can produce signaling molecules with distinct or antagonistic functions and localization to different subcellular compartments. This newly discovered link expands our understanding of the biology of immune and neuroendocrine cells, and has the potential to open new windows of opportunity for treatment of neurodegenerative disorders. PMID:26371053

  4. Splicing Regulation of Pro-Inflammatory Cytokines and Chemokines: At the Interface of the Neuroendocrine and Immune Systems

    PubMed Central

    Shakola, Felitsiya; Suri, Parul; Ruggiu, Matteo

    2015-01-01

    Alternative splicing plays a key role in posttranscriptional regulation of gene expression, allowing a single gene to encode multiple protein isoforms. As such, alternative splicing amplifies the coding capacity of the genome enormously, generates protein diversity, and alters protein function. More than 90% of human genes undergo alternative splicing, and alternative splicing is especially prevalent in the nervous and immune systems, tissues where cells need to react swiftly and adapt to changes in the environment through carefully regulated mechanisms of cell differentiation, migration, targeting, and activation. Given its prevalence and complexity, this highly regulated mode of gene expression is prone to be affected by disease. In the following review, we look at how alternative splicing of signaling molecules—cytokines and their receptors—changes in different pathological conditions, from chronic inflammation to neurologic disorders, providing means of functional interaction between the immune and neuroendocrine systems. Switches in alternative splicing patterns can be very dynamic and can produce signaling molecules with distinct or antagonistic functions and localization to different subcellular compartments. This newly discovered link expands our understanding of the biology of immune and neuroendocrine cells, and has the potential to open new windows of opportunity for treatment of neurodegenerative disorders. PMID:26371053

  5. The ZZ domain of dystrophin in DMD: making sense of missense mutations.

    PubMed

    Vulin, Adeline; Wein, Nicolas; Strandjord, Dana M; Johnson, Eric K; Findlay, Andrew R; Maiti, Baijayanta; Howard, Michael T; Kaminoh, Yuuki J; Taylor, Laura E; Simmons, Tabatha R; Ray, Will C; Montanaro, Federica; Ervasti, Jim M; Flanigan, Kevin M

    2014-02-01

    Duchenne muscular dystrophy (DMD) is associated with the loss of dystrophin, which plays an important role in myofiber integrity via interactions with β-dystroglycan and other members of the transmembrane dystrophin-associated protein complex. The ZZ domain, a cysteine-rich zinc-finger domain near the dystrophin C-terminus, is implicated in forming a stable interaction between dystrophin and β-dystroglycan, but the mechanism of pathogenesis of ZZ missense mutations has remained unclear because not all such mutations have been shown to alter β-dystroglycan binding in previous experimental systems. We engineered three ZZ mutations (p.Cys3313Phe, p.Asp3335His, and p.Cys3340Tyr) into a short construct similar to the Dp71 dystrophin isoform for in vitro and in vivo studies and delineated their effect on protein expression, folding properties, and binding partners. Our results demonstrate two distinct pathogenic mechanisms for ZZ missense mutations. The cysteine mutations result in diminished or absent subsarcolemmal expression because of protein instability, likely due to misfolding. In contrast, the aspartic acid mutation disrupts binding with β-dystroglycan despite an almost normal expression at the membrane, confirming a role for the ZZ domain in β-dystroglycan binding but surprisingly demonstrating that such binding is not required for subsarcolemmal localization of dystrophin, even in the absence of actin binding domains. PMID:24302611

  6. Splicing fidelity

    PubMed Central

    Koodathingal, Prakash; Staley, Jonathan P.

    2013-01-01

    The spliceosome discriminates against suboptimal substrates, both during assembly and catalysis, thereby enhancing specificity during pre-mRNA splicing. Central to such fidelity mechanisms are a conserved subset of the DEAD- and DEAH-box ATPases, which belong to a superfamily of proteins that mediate RNP rearrangements in almost all RNA-dependent processes in the cell. Through an investigation of the mechanisms contributing to the specificity of 5′ splice site cleavage, two related reports, one from our lab and the other from the Cheng lab, have provided insights into fidelity mechanisms utilized by the spliceosome. In our work, we found evidence for a kinetic proofreading mechanism in splicing in which the DEAH-box ATPase Prp16 discriminates against substrates undergoing slow 5′ splice site cleavage. Additionally, our study revealed that discriminated substrates are discarded through a general spliceosome disassembly pathway, mediated by another DEAH-box ATPase Prp43. In their work, Tseng et al. described the underlying molecular events through which Prp16 discriminates against a splicing substrate during 5′ splice site cleavage. Here, we present a synthesis of these two studies and, additionally, provide the first biochemical evidence for discrimination of a suboptimal splicing substrate just prior to 5′ splice site cleavage. Together, these findings support a general mechanism for a ubiquitous superfamily of ATPases in enhancing specificity during RNA-dependent processes in the cell. PMID:23770752

  7. Molecular dissection of step 2 catalysis of yeast pre-mRNA splicing investigated in a purified system.

    PubMed

    Ohrt, Thomas; Odenwälder, Peter; Dannenberg, Julia; Prior, Mira; Warkocki, Zbigniew; Schmitzová, Jana; Karaduman, Ramazan; Gregor, Ingo; Enderlein, Jörg; Fabrizio, Patrizia; Lührmann, Reinhard

    2013-07-01

    Step 2 catalysis of pre-mRNA splicing entails the excision of the intron and ligation of the 5' and 3' exons. The tasks of the splicing factors Prp16, Slu7, Prp18, and Prp22 in the formation of the step 2 active site of the spliceosome and in exon ligation, and the timing of their recruitment, remain poorly understood. Using a purified yeast in vitro splicing system, we show that only the DEAH-box ATPase Prp16 is required for formation of a functional step 2 active site and for exon ligation. Efficient docking of the 3' splice site (3'SS) to the active site requires only Slu7/Prp18 but not Prp22. Spliceosome remodeling by Prp16 appears to be subtle as only the step 1 factor Cwc25 is dissociated prior to step 2 catalysis, with its release dependent on docking of the 3'SS to the active site and Prp16 action. We show by fluorescence cross-correlation spectroscopy that Slu7/Prp18 and Prp16 bind early to distinct, low-affinity binding sites on the step-1-activated B* spliceosome, which are subsequently converted into high-affinity sites. Our results shed new light on the factor requirements for step 2 catalysis and the dynamics of step 1 and 2 factors during the catalytic steps of splicing. PMID:23685439

  8. Deletion of Dystrophin In-Frame Exon 5 Leads to a Severe Phenotype: Guidance for Exon Skipping Strategies

    PubMed Central

    Toh, Zhi Yon Charles; Thandar Aung-Htut, May; Pinniger, Gavin; Adams, Abbie M.; Krishnaswarmy, Sudarsan; Wong, Brenda L.; Fletcher, Sue; Wilton, Steve D.

    2016-01-01

    Duchenne and Becker muscular dystrophy severity depends upon the nature and location of the DMD gene lesion and generally correlates with the dystrophin open reading frame. However, there are striking exceptions where an in-frame genomic deletion leads to severe pathology or protein-truncating mutations (nonsense or frame-shifting indels) manifest as mild disease. Exceptions to the dystrophin reading frame rule are usually resolved after molecular diagnosis on muscle RNA. We report a moderate/severe Becker muscular dystrophy patient with an in-frame genomic deletion of DMD exon 5. This mutation has been reported by others as resulting in Duchenne or Intermediate muscular dystrophy, and the loss of this in-frame exon in one patient led to multiple splicing events, including omission of exon 6, that disrupts the open reading frame and is consistent with a severe phenotype. The patient described has a deletion of dystrophin exon 5 that does not compromise recognition of exon 6, and although the deletion does not disrupt the reading frame, his clinical presentation is more severe than would be expected for classical Becker muscular dystrophy. We suggest that the dystrophin isoform lacking the actin-binding sequence encoded by exon 5 is compromised, reflected by the phenotype resulting from induction of this dystrophin isoform in mouse muscle in vivo. Hence, exon skipping to address DMD-causing mutations within DMD exon 5 may not yield an isoform that confers marked clinical benefit. Additional studies will be required to determine whether multi-exon skipping strategies could yield more functional dystrophin isoforms, since some BMD patients with larger in-frame deletions in this region have been reported with mild phenotypes. PMID:26745801

  9. Deletion of Dystrophin In-Frame Exon 5 Leads to a Severe Phenotype: Guidance for Exon Skipping Strategies.

    PubMed

    Toh, Zhi Yon Charles; Thandar Aung-Htut, May; Pinniger, Gavin; Adams, Abbie M; Krishnaswarmy, Sudarsan; Wong, Brenda L; Fletcher, Sue; Wilton, Steve D

    2016-01-01

    Duchenne and Becker muscular dystrophy severity depends upon the nature and location of the DMD gene lesion and generally correlates with the dystrophin open reading frame. However, there are striking exceptions where an in-frame genomic deletion leads to severe pathology or protein-truncating mutations (nonsense or frame-shifting indels) manifest as mild disease. Exceptions to the dystrophin reading frame rule are usually resolved after molecular diagnosis on muscle RNA. We report a moderate/severe Becker muscular dystrophy patient with an in-frame genomic deletion of DMD exon 5. This mutation has been reported by others as resulting in Duchenne or Intermediate muscular dystrophy, and the loss of this in-frame exon in one patient led to multiple splicing events, including omission of exon 6, that disrupts the open reading frame and is consistent with a severe phenotype. The patient described has a deletion of dystrophin exon 5 that does not compromise recognition of exon 6, and although the deletion does not disrupt the reading frame, his clinical presentation is more severe than would be expected for classical Becker muscular dystrophy. We suggest that the dystrophin isoform lacking the actin-binding sequence encoded by exon 5 is compromised, reflected by the phenotype resulting from induction of this dystrophin isoform in mouse muscle in vivo. Hence, exon skipping to address DMD-causing mutations within DMD exon 5 may not yield an isoform that confers marked clinical benefit. Additional studies will be required to determine whether multi-exon skipping strategies could yield more functional dystrophin isoforms, since some BMD patients with larger in-frame deletions in this region have been reported with mild phenotypes. PMID:26745801

  10. Rapid generation of splicing reporters with pSpliceExpress

    PubMed Central

    Kishore, Shivendra; Khanna, Amit; Stamm, Stefan

    2008-01-01

    Almost all human protein-coding transcripts undergo pre-mRNA splicing and a majority of them is alternatively spliced. The most common technique used to analyze the regulation of an alternative exon is through reporter minigene constructs. However, their construction is time-consuming and is often complicated by the limited availability of appropriate restriction sites. Here, we report a fast and simple recombination-based method to generate splicing reporter genes, using a new vector, pSpliceExpress. The system allows generation of minigenes within one week. Minigenes generated with pSpliceExpress show the same regulation as displayed by conventionally cloned reporter constructs and provide an alternate avenue to study splice site selection in vivo. PMID:18930792

  11. Mini-dystrophin Expression Down-regulates IP3-mediated Calcium Release Events in Resting Dystrophin-deficient Muscle Cells

    PubMed Central

    Balghi, Haouaria; Sebille, Stéphane; Mondin, Ludivine; Cantereau, Anne; Constantin, Bruno; Raymond, Guy; Cognard, Christian

    2006-01-01

    We present here evidence for the enhancement, at rest, of an inositol 1,4,5-trisphosphate (IP3)–mediated calcium signaling pathway in myotubes from dystrophin-deficient cell lines (SolC1(−)) as compared to a cell line from the same origin but transfected with mini-dystrophin (SolD(+)). With confocal microscopy, the number of sites discharging calcium (release site density [RSD]) was quantified and found more elevated in SolC1(−) than in SolD(+) myotubes. Variations of membrane potential had no significant effect on this difference, and higher resting [Ca2+]i in SolC1(−) (Marchand, E., B. Constantin, H. Balghi, M.C. Claudepierre, A. Cantereau, C. Magaud, A. Mouzou, G. Raymond, S. Braun, and C. Cognard. 2004. Exp. Cell Res. 297:363–379) cannot explain alone higher RSD. The exposure with SR Ca2+ channel inhibitors (ryanodine and 2-APB) and phospholipase C inhibitor (U73122) significantly reduced RSD in both cell types but with a stronger effect in dystrophin-deficient SolC1(−) myotubes. Immunocytochemistry allowed us to localize ryanodine receptors (RyRs) as well as IP3 receptors (IP3Rs), IP3R-1 and IP3R-2 isoforms, indicating the presence of both RyRs-dependent and IP3-dependent release systems in both cells. We previously reported evidence for the enhancement, through a Gi protein, of the IP3-mediated calcium signaling pathway in SolC1(−) as compared to SolD(+) myotubes during a high K+ stimulation (Balghi, H., S. Sebille, B. Constantin, S. Patri, V. Thoreau, L. Mondin, E. Mok, A. Kitzis, G. Raymond, and C. Cognard. 2006. J. Gen. Physiol. 127:171–182). Here we show that, at rest, these regulation mechanisms are also involved in the modulation of calcium release activities. The enhancement of resting release activity may participate in the calcium overload observed in dystrophin-deficient myotubes, and our findings support the hypothesis of the regulatory role of mini-dystrophin on intracellular signaling. PMID:16847098

  12. Adhalin, the 50 kD dystrophin associated protein, is not the locus for severe childhood autosomal recessive dystrophy (SCARMD)

    SciTech Connect

    McNally, E.M.; Selig, S.; Kunkel, L.M.

    1994-09-01

    Mutations in the carboxyl-terminus in dystrophin are normally sufficient to produce severely dystrophic muscle. This portion of dystrophin binds a complex of dystrophin-associated glycoproteins (DAGs). The genes encoding these DAGs are candidate genes for causing neuromuscular disease. Immunoreactivity for adhalin, the 50 kD DAG, is absent in muscle biopsies from patients with SCARMD, a form of dystrophy clinically similar Duchenne muscular dystrophy. Prior linkage analysis in SCARMD families revealed that the disease gene segregates with markers on chromosome 13. To determine the molecular role that adhalin may play in SCARMD, human cDNA and genomic sequences were isolated. Primers were designed based on predicted areas of conservation in rabbit adhalin and used in RT-PCR with human skeletal and cardiac muscle. RT-PCR products were confirmed by sequence as human adhalin and then used as probes for screening human cDNA and genomic libraries. Human and rabbit adhalin are 90% identical, and among the cDNAs, a novel splice form of adhalin was seen which may encode part of the 35 kD component of the dystrophin-glycoprotein complex. To our surprise, only human/rodent hybrids containing human chromosome 17 amplified adhalin sequences in a PCR analysis. FISH analysis with three overlapping genomic sequences confirmed the chromosome 17 location and further delineated the map position to 17q21. Therefore, adhalin is excluded as the gene causing SCARMD.

  13. Combination Antisense Treatment for Destructive Exon Skipping of Myostatin and Open Reading Frame Rescue of Dystrophin in Neonatal mdx Mice.

    PubMed

    Lu-Nguyen, Ngoc B; Jarmin, Susan A; Saleh, Amer F; Popplewell, Linda; Gait, Michael J; Dickson, George

    2015-08-01

    The fatal X-linked Duchenne muscular dystrophy (DMD), characterized by progressive muscle wasting and muscle weakness, is caused by mutations within the DMD gene. The use of antisense oligonucleotides (AOs) modulating pre-mRNA splicing to restore the disrupted dystrophin reading frame, subsequently generating a shortened but functional protein has emerged as a potential strategy in DMD treatment. AO therapy has recently been applied to induce out-of-frame exon skipping of myostatin pre-mRNA, knocking-down expression of myostatin protein, and such an approach is suggested to enhance muscle hypertrophy/hyperplasia and to reduce muscle necrosis. Within this study, we investigated dual exon skipping of dystrophin and myostatin pre-mRNAs using phosphorodiamidate morpholino oligomers conjugated with an arginine-rich peptide (B-PMOs). Intraperitoneal administration of B-PMOs was performed in neonatal mdx males on the day of birth, and at weeks 3 and 6. At week 9, we observed in treated mice (as compared to age-matched, saline-injected controls) normalization of muscle mass, a recovery in dystrophin expression, and a decrease in muscle necrosis, particularly in the diaphragm. Our data provide a proof of concept for antisense therapy combining dystrophin restoration and myostatin inhibition for the treatment of DMD. PMID:25959011

  14. Combination antisense treatment for destructive exon skipping of myostatin and open reading frame rescue of dystrophin in neonatal mdx mice

    PubMed Central

    Lu-Nguyen, Ngoc B.; Jarmin, Susan A.; Saleh, Amer F.; Popplewell, Linda; Gait, Michael J.; Dickson, George

    2015-01-01

    The fatal X-linked Duchenne muscular dystrophy (DMD), characterized by progressive muscle wasting and muscle weakness, is caused by mutations within the DMD gene. The use of antisense oligonucleotides (AOs) modulating pre-mRNA splicing to restore the disrupted dystrophin reading frame, subsequently generating a shortened but functional protein has emerged as a potential strategy in DMD treatment. AO therapy has recently been applied to induce out-of-frame exon skipping of myostatin pre-mRNA, knocking-down expression of myostatin protein, and such an approach is suggested to enhance muscle hypertrophy/hyperplasia and to reduce muscle necrosis. Within this study, we investigated dual exon skipping of dystrophin and myostatin pre-mRNAs using phosphorodiamidate morpholino oligomers conjugated with an arginine-rich peptide (B-PMOs). Intraperitoneal administration of B-PMOs was performed in neonatal mdx males on the day of birth, and at weeks 3 and 6. At week 9, we observed in treated mice (as compared to age-matched, saline-injected controls) normalization of muscle mass, a recovery in dystrophin expression, and a decrease in muscle necrosis, particularly in the diaphragm. Our data provide a proof of concept for antisense therapy combining dystrophin restoration and myostatin inhibition for the treatment of DMD. PMID:25959011

  15. Revisiting the dystrophin-ATP connection: How half a century of research still implicates mitochondrial dysfunction in Duchenne Muscular Dystrophy aetiology.

    PubMed

    Timpani, Cara A; Hayes, Alan; Rybalka, Emma

    2015-12-01

    Duchenne Muscular Dystrophy (DMD) is a fatal neuromuscular disease that is characterised by dystrophin-deficiency and chronic Ca(2+)-induced skeletal muscle wasting, which currently has no cure. DMD was once considered predominantly as a metabolic disease due to the myriad of metabolic insufficiencies evident in the musculature, however this aspect of the disease has been extensively ignored since the discovery of dystrophin. The collective historical and contemporary literature documenting these metabolic nuances has culminated in a series of studies that importantly demonstrate that metabolic dysfunction exists independent of dystrophin expression and a mild disease phenotype can be expressed even in the complete absence of dystrophin expression. Targeting and supporting metabolic pathways with anaplerotic and other energy-enhancing supplements has also shown therapeutic value. We explore the hypothesis that DMD is characterised by a systemic mitochondrial impairment that is central to disease aetiology rather than a secondary pathophysiological consequence of dystrophin-deficiency. PMID:26365249

  16. Interplay between DMD point mutations and splicing signals in Dystrophinopathy phenotypes.

    PubMed

    Juan-Mateu, Jonàs; González-Quereda, Lidia; Rodríguez, Maria José; Verdura, Edgard; Lázaro, Kira; Jou, Cristina; Nascimento, Andrés; Jiménez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jesús; Baiget, Montserrat; Gallano, Pia

    2013-01-01

    DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements. PMID:23536893

  17. Interplay between DMD Point Mutations and Splicing Signals in Dystrophinopathy Phenotypes

    PubMed Central

    Juan-Mateu, Jonàs; González-Quereda, Lidia; Rodríguez, Maria José; Verdura, Edgard; Lázaro, Kira; Jou, Cristina; Nascimento, Andrés; Jiménez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jesús; Baiget, Montserrat; Gallano, Pia

    2013-01-01

    DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements. PMID:23536893

  18. Tissue distribution of the dystrophin-related gene product and expression in the mdx and dy mouse

    SciTech Connect

    Love, D.R.; Marsden, R.F.; Bloomfield, J.F.; Davies, K.E. ); Morris, G.E.; Ellis, J.M. ); Fairbrother, U.; Edwards, Y.H. ); Slater, C.P. ); Parry, D.J. )

    1991-04-15

    The authors have previously reported a dystrophin-related locus (DMDL for Duchenne muscular dystrophy-like) on human chromosome 6 that maps close to the dy mutation on mouse chromosome 10. Here they show that this gene is expressed in a wide range of tissues at varying levels. The transcript is particularly abundant in several human fetal tissues, including heart, placenta, and intestine. Studies with antisera raised against a DMDL fusion protein identify a 400,000 M{sub r} protein in all mouse tissues tested, including those of mdx and dy mice. Unlike the dystrophin gene, the DMDL gene transcript is not differentially spliced at the 3{prime} end in either fetal muscle or brain.

  19. Permeable trypanosome cells as a model system for transcription and trans-splicing.

    PubMed Central

    Ullu, E; Tschudi, C

    1990-01-01

    We have established conditions for Trypanosoma brucei permeable cells to study transcription and trans-splicing. We found that the concentration of monovalent and, to a lesser extent, divalent ions plays a critical role for the expression of a number of different genes. Most remarkably, the synthesis of the spliced leader (SL) RNA was optimal at 20 mM KCl, whereas higher potassium concentrations were inhibitory. In addition, MgCl2 concentrations above 3 mM led to the accumulation of a 3' end shortened SL RNA species, which has been previously reported not to participate in trans-splicing. Using conditions optimal for the synthesis of the SL RNA, we observed accurate trans-splicing of newly-synthesized alpha-tubulin RNA. Moreover, we detected the SL intron both joined to high molecular weight RNAs in the form of branched Y-structures and as a free linear molecule, which rapidly turned over. Furthermore, ionic concentrations that inhibit the synthesis of the SL RNA produced exclusively unspliced alpha-tubulin RNA, thus demonstrating that transcription and trans-splicing can be uncoupled. Images PMID:2356121

  20. Effective Dystrophin Restoration by a Novel Muscle-Homing Peptide–Morpholino Conjugate in Dystrophin-Deficient mdx Mice

    PubMed Central

    Gao, Xianjun; Zhao, Jingwen; Han, Gang; Zhang, Yajie; Dong, Xue; Cao, Limin; Wang, Qingsong; Moulton, Hong M; Yin, HaiFang

    2014-01-01

    Antisense oligonucleotide (AO)–mediated splice correction therapy for Duchenne muscular dystrophy has shown huge promise from recent phase 2b clinical trials, however high doses and costs are required and targeted delivery can lower both of these. We have previously demonstrated the feasibility of targeted delivery of AOs by conjugating a chimeric peptide, consisting of a muscle-specific peptide and a cell-penetrating peptide, to AOs in mdx mice. Although increased uptake in muscle was observed, the majority of peptide–AO conjugate was found in the liver. To search for more effective muscle-homing peptides, we carried out in vitro biopanning in myoblasts and identified a novel 12-mer peptide (M12) showing preferential binding to skeletal muscle compared to the liver. When conjugated to phosphorodiamidate morpholino oligomers, ~25% of normal level of dystrophin expression was achieved in body-wide skeletal muscles in mdx mice with significant recovery in grip strength, whereas <2% in corresponding tissues treated with either muscle-specific peptide–phosphorodiamidate morpholino oligomer or unmodified phosphorodiamidate morpholino oligomer under identical conditions. Our data provide evidences for the first time that a muscle-homing peptide alone can enhance AO delivery to muscle without appreciable toxicity at 75 mg/kg, suggesting M12-phosphorodiamidate morpholino oligomer can be an alternative option to current AOs. PMID:24732757

  1. Neuron-specific splicing of the Alzheimer amyloid precursor protein gene in a mini-gene system.

    PubMed

    Yamada, T; Goto, I; Sakaki, Y

    1993-08-31

    Several forms of Alzheimer amyloid precursor protein (APP) mRNA are generated by alternative splicing. Among them, the APP695 mRNA skipping the exon 7 and 8 is expressed specifically in neurons, suggesting that this alternative splicing is regulated in a neuron-specific manner. As the first step for investigating the mechanism of the neuron-specific splicing, a mini-gene system was developed, in which mini-APP genes consisting of the exon 6, 7, 8, 9 and their flanking regions were introduced into neuronal and nonneuronal cultured cell lines to see their expression profiles. In the system the exon 7 and 8 of the mini-gene were significantly skipped in the neuronal cell, and the deletion study indicated that cis-acting elements for skipping the exons existed in the corresponding skipped-exon and its flanking regions. A small deletion upstream of the exon 8 suppressed the skipping of the exon 8 in the neuronal cell, suggesting that one of the regulatory sequence(s) for the exon skipping exists in a small region upstream of the skipped exon. PMID:8363619

  2. Development of Therapeutic Splice-Switching Oligonucleotides

    PubMed Central

    Kryczka, Adrianna; Liu, Yuqi; Badi, Yusef E.; Wong, Jessie J.; Owen, James S.; Khoo, Bernard

    2014-01-01

    Abstract Synthetic splice-switching oligonucleotides (SSOs) target nuclear pre-mRNA molecules to change exon splicing and generate an alternative protein isoform. Clinical trials with two competitive SSO drugs are underway to treat Duchenne muscular dystrophy (DMD). Beyond DMD, many additional therapeutic applications are possible, with some in phase 1 clinical trials or advanced preclinical evaluation. Here, we present an overview of the central factors involved in developing therapeutic SSOs for the treatment of diseases. The selection of susceptible pre-mRNA target sequences, as well as the design and chemical modification of SSOs to increase SSO stability and effectiveness, are key initial considerations. Identification of effective SSO target sequences is still largely empirical and published guidelines are not a universal guarantee for success. Specifically, exon-targeted SSOs, which are successful in modifying dystrophin splicing, can be ineffective for splice-switching in other contexts. Chemical modifications, importantly, are associated with certain characteristic toxicities, which need to be addressed as target diseases require chronic treatment with SSOs. Moreover, SSO delivery in adequate quantities to the nucleus of target cells without toxicity can prove difficult. Last, the means by which these SSOs are administered needs to be acceptable to the patient. Engineering an efficient therapeutic SSO, therefore, necessarily entails a compromise between desirable qualities and effectiveness. Here, we describe how the application of optimal solutions may differ from case to case. PMID:24826963

  3. Dystrophin: The dead calm of a dogma.

    PubMed

    Górecki, Dariusz C

    2016-01-01

    Duchenne muscular dystrophy (DMD) is the most common inherited muscle disease leading to severe disability and death of young men. Current interventions are palliative as no treatment improves the long-term outcome. Therefore, new therapeutic modalities with translational potential are urgently needed and abnormalities downstream from the absence of dystrophin are realistic targets. It has been shown that DMD mutations alter extracellular ATP (eATP) signaling via P2RX7 purinoceptor upregulation, which leads to autophagic death of dystrophic muscle cells. Furthermore, the eATP-P2RX7 axis contributes to DMD pathology by stimulating harmful inflammatory responses. We demonstrated recently that genetic ablation or pharmacological inhibition of P2RX7 in the mdx mouse model of DMD produced functional attenuation of both muscle and non-muscle symptoms, establishing this receptor as an attractive therapeutic target. Central to the argument presented here, this purinergic phenotype affects dystrophic myoblasts. Muscle cells were believed not to be affected at this stage of differentiation, as they do not produce detectable dystrophin protein. Our findings contradict the central hypothesis stating that aberrant dystrophin expression is inconsequential in myoblasts and the DMD pathology results from effects such as sarcolemma fragility, due to the absence of dystrophin, in differentiated myofibres. However, we discuss here the evidence that, already in myogenic cells, DMD mutations produce a plethora of abnormalities, including in cell proliferation, differentiation, energy metabolism, Ca(2+) homeostasis and death, leading to impaired muscle regeneration. We hope that this discussion may bring to light further results that will help re-evaluating the established belief. Clearly, understanding how DMD mutations alter such a range of functions in myogenic cells is vital for developing effective therapies. PMID:27141413

  4. Dystrophin: The dead calm of a dogma

    PubMed Central

    Górecki, Dariusz C.

    2016-01-01

    ABSTRACT Duchenne muscular dystrophy (DMD) is the most common inherited muscle disease leading to severe disability and death of young men. Current interventions are palliative as no treatment improves the long-term outcome. Therefore, new therapeutic modalities with translational potential are urgently needed and abnormalities downstream from the absence of dystrophin are realistic targets. It has been shown that DMD mutations alter extracellular ATP (eATP) signaling via P2RX7 purinoceptor upregulation, which leads to autophagic death of dystrophic muscle cells. Furthermore, the eATP-P2RX7 axis contributes to DMD pathology by stimulating harmful inflammatory responses. We demonstrated recently that genetic ablation or pharmacological inhibition of P2RX7 in the mdx mouse model of DMD produced functional attenuation of both muscle and non-muscle symptoms, establishing this receptor as an attractive therapeutic target. Central to the argument presented here, this purinergic phenotype affects dystrophic myoblasts. Muscle cells were believed not to be affected at this stage of differentiation, as they do not produce detectable dystrophin protein. Our findings contradict the central hypothesis stating that aberrant dystrophin expression is inconsequential in myoblasts and the DMD pathology results from effects such as sarcolemma fragility, due to the absence of dystrophin, in differentiated myofibres. However, we discuss here the evidence that, already in myogenic cells, DMD mutations produce a plethora of abnormalities, including in cell proliferation, differentiation, energy metabolism, Ca2+ homeostasis and death, leading to impaired muscle regeneration. We hope that this discussion may bring to light further results that will help re-evaluating the established belief. Clearly, understanding how DMD mutations alter such a range of functions in myogenic cells is vital for developing effective therapies. PMID:27141413

  5. Dystrophin delivery in dystrophin-deficient DMDmdx skeletal muscle by isogenic muscle-derived stem cell transplantation.

    PubMed

    Ikezawa, Makoto; Cao, Baohong; Qu, Zhuqing; Peng, Hairong; Xiao, Xiao; Pruchnic, Ryan; Kimura, Shigemi; Miike, Teruhisa; Huard, Johnny

    2003-11-01

    Duchenne's muscular dystrophy (DMD) is a lethal muscle disease caused by a lack of dystrophin expression at the sarcolemma of muscle fibers. We investigated retroviral vector delivery of dystrophin in dystrophin-deficient DMD(mdx) (hereafter referred to as mdx) mice via an ex vivo approach using mdx muscle-derived stem cells (MDSCs). We generated a retrovirus carrying a functional human mini-dystrophin (RetroDys3999) and used it to stably transduce mdx MDSCs obtained by the preplate technique (MD3999). These MD3999 cells expressed dystrophin and continued to express stem cell markers, including CD34 and Sca-1. MD3999 cells injected into mdx mouse skeletal muscle were able to deliver dystrophin. Though a relatively low number of dystrophin-positive myofibers was generated within the gastrocnemius muscle, these fibers persisted for up to 24 weeks postinjection. The injection of cells from additional MDSC/Dys3999 clones into mdx skeletal muscle resulted in varying numbers of dystrophin-positive myofibers, suggesting a differential regenerating capacity among the clones. At 2 and 4 weeks postinjection, the infiltration of CD4- and CD8-positive lymphocytes and a variety of cytokines was detected within the injected site. These data suggest that the transplantation of retrovirally transduced mdx MDSCs can enable persistent dystrophin restoration in mdx skeletal muscle; however, the differential regenerating capacity observed among the MDSC/Dys3999 clones and the postinjection immune response are potential challenges facing this technology. PMID:14577915

  6. Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy.

    PubMed

    Ousterout, David G; Kabadi, Ami M; Thakore, Pratiksha I; Majoros, William H; Reddy, Timothy E; Gersbach, Charles A

    2015-01-01

    The CRISPR/Cas9 genome-editing platform is a promising technology to correct the genetic basis of hereditary diseases. The versatility, efficiency and multiplexing capabilities of the CRISPR/Cas9 system enable a variety of otherwise challenging gene correction strategies. Here, we use the CRISPR/Cas9 system to restore the expression of the dystrophin gene in cells carrying dystrophin mutations that cause Duchenne muscular dystrophy (DMD). We design single or multiplexed sgRNAs to restore the dystrophin reading frame by targeting the mutational hotspot at exons 45-55 and introducing shifts within exons or deleting one or more exons. Following gene editing in DMD patient myoblasts, dystrophin expression is restored in vitro. Human dystrophin is also detected in vivo after transplantation of genetically corrected patient cells into immunodeficient mice. Importantly, the unique multiplex gene-editing capabilities of the CRISPR/Cas9 system facilitate the generation of a single large deletion that can correct up to 62% of DMD mutations. PMID:25692716

  7. Multiplex CRISPR/Cas9-Based Genome Editing for Correction of Dystrophin Mutations that Cause Duchenne Muscular Dystrophy

    PubMed Central

    Ousterout, David G.; Kabadi, Ami M.; Thakore, Pratiksha I.; Majoros, William H.; Reddy, Timothy E.; Gersbach, Charles A.

    2015-01-01

    The CRISPR/Cas9 genome editing platform is a promising technology to correct the genetic basis of hereditary diseases. The versatility, efficiency, and multiplexing capabilities of the CRISPR/Cas9 system enable a variety of otherwise challenging gene correction strategies. Here we use the CRISPR/Cas9 system to restore the expression of the dystrophin gene in cells carrying dystrophin mutations that cause Duchenne muscular dystrophy (DMD). We design single or multiplexed sgRNAs to restore the dystrophin reading frame by targeting the mutational hotspot at exons 45–55 and introducing shifts within exons or deleting one or more exons. Following gene editing in DMD patient myoblasts, dystrophin expression is restored in vitro. Human dystrophin is also detected in vivo after transplantation of genetically corrected patient cells into immunodeficient mice. Importantly, the unique multiplex gene editing capabilities of the CRISPR/Cas9 system facilitate the generation of a single large deletion that can correct up to 62% of DMD mutations. PMID:25692716

  8. Laryngeal Muscles Are Spared in the Dystrophin Deficient "mdx" Mouse

    ERIC Educational Resources Information Center

    Thomas, Lisa B.; Joseph, Gayle L.; Adkins, Tracey D.; Andrade, Francisco H.; Stemple, Joseph C.

    2008-01-01

    Purpose: "Duchenne muscular dystrophy (DMD)" is caused by the loss of the cytoskeletal protein, dystrophin. The disease leads to severe and progressive skeletal muscle wasting. Interestingly, the disease spares some muscles. The purpose of the study was to determine the effects of dystrophin deficiency on 2 intrinsic laryngeal muscles, the…

  9. Disease-proportional proteasomal degradation of missense dystrophins

    PubMed Central

    Talsness, Dana M.; Belanto, Joseph J.; Ervasti, James M.

    2015-01-01

    The 427-kDa protein dystrophin is expressed in striated muscle where it physically links the interior of muscle fibers to the extracellular matrix. A range of mutations in the DMD gene encoding dystrophin lead to a severe muscular dystrophy known as Duchenne (DMD) or a typically milder form known as Becker (BMD). Patients with nonsense mutations in dystrophin are specifically targeted by stop codon read-through drugs, whereas out-of-frame deletions and insertions are targeted by exon-skipping therapies. Both treatment strategies are currently in clinical trials. Dystrophin missense mutations, however, cause a wide range of phenotypic severity in patients. The molecular and cellular consequences of such mutations are not well understood, and there are no therapies specifically targeting this genotype. Here, we have modeled two representative missense mutations, L54R and L172H, causing DMD and BMD, respectively, in full-length dystrophin. In vitro, the mutation associated with the mild phenotype (L172H) caused a minor decrease in tertiary stability, whereas the L54R mutation associated with a severe phenotype had a more dramatic effect. When stably expressed in mammalian muscle cells, the mutations caused steady-state decreases in dystrophin protein levels inversely proportional to the tertiary stability and directly caused by proteasomal degradation. Both proteasome inhibitors and heat shock activators were able to increase mutant dystrophin to WT levels, establishing the new cell lines as a platform to screen for potential therapeutics personalized to patients with destabilized dystrophin. PMID:26392559

  10. Disease-proportional proteasomal degradation of missense dystrophins.

    PubMed

    Talsness, Dana M; Belanto, Joseph J; Ervasti, James M

    2015-10-01

    The 427-kDa protein dystrophin is expressed in striated muscle where it physically links the interior of muscle fibers to the extracellular matrix. A range of mutations in the DMD gene encoding dystrophin lead to a severe muscular dystrophy known as Duchenne (DMD) or a typically milder form known as Becker (BMD). Patients with nonsense mutations in dystrophin are specifically targeted by stop codon read-through drugs, whereas out-of-frame deletions and insertions are targeted by exon-skipping therapies. Both treatment strategies are currently in clinical trials. Dystrophin missense mutations, however, cause a wide range of phenotypic severity in patients. The molecular and cellular consequences of such mutations are not well understood, and there are no therapies specifically targeting this genotype. Here, we have modeled two representative missense mutations, L54R and L172H, causing DMD and BMD, respectively, in full-length dystrophin. In vitro, the mutation associated with the mild phenotype (L172H) caused a minor decrease in tertiary stability, whereas the L54R mutation associated with a severe phenotype had a more dramatic effect. When stably expressed in mammalian muscle cells, the mutations caused steady-state decreases in dystrophin protein levels inversely proportional to the tertiary stability and directly caused by proteasomal degradation. Both proteasome inhibitors and heat shock activators were able to increase mutant dystrophin to WT levels, establishing the new cell lines as a platform to screen for potential therapeutics personalized to patients with destabilized dystrophin. PMID:26392559

  11. Splicing Programs and Cancer

    PubMed Central

    Germann, Sophie; Gratadou, Lise; Dutertre, Martin; Auboeuf, Didier

    2012-01-01

    Numerous studies report splicing alterations in a multitude of cancers by using gene-by-gene analysis. However, understanding of the role of alternative splicing in cancer is now reaching a new level, thanks to the use of novel technologies allowing the analysis of splicing at a large-scale level. Genome-wide analyses of alternative splicing indicate that splicing alterations can affect the products of gene networks involved in key cellular programs. In addition, many splicing variants identified as being misregulated in cancer are expressed in normal tissues. These observations suggest that splicing programs contribute to specific cellular programs that are altered during cancer initiation and progression. Supporting this model, recent studies have identified splicing factors controlling cancer-associated splicing programs. The characterization of splicing programs and their regulation by splicing factors will allow a better understanding of the genetic mechanisms involved in cancer initiation and progression and the development of new therapeutic targets. PMID:22132318

  12. Extensive and prolonged restoration of dystrophin expression with vivo-morpholino-mediated multiple exon skipping in dystrophic dogs.

    PubMed

    Yokota, Toshifumi; Nakamura, Akinori; Nagata, Tetsuya; Saito, Takashi; Kobayashi, Masanori; Aoki, Yoshitsugu; Echigoya, Yusuke; Partridge, Terence; Hoffman, Eric P; Takeda, Shin'ichi

    2012-10-01

    Duchenne muscular dystrophy (DMD) is a severe and the most prevalent form of muscular dystrophy, characterized by rapid progression of muscle degeneration. Antisense-mediated exon skipping is currently one of the most promising therapeutic options for DMD. However, unmodified antisense oligos such as morpholinos require frequent (weekly or bi-weekly) injections. Recently, new generation morpholinos such as vivo-morpholinos are reported to lead to extensive and prolonged dystrophin expression in the dystrophic mdx mouse, an animal model of DMD. The vivo-morpholino contains a cell-penetrating moiety, octa-guanidine dendrimer. Here, we sought to test the efficacy of multiple exon skipping of exons 6-8 with vivo-morpholinos in the canine X-linked muscular dystrophy, which harbors a splice site mutation at the boundary of intron 6 and exon 7. We designed and optimized novel antisense cocktail sequences and combinations for exon 8 skipping and demonstrated effective exon skipping in dystrophic dogs in vivo. Intramuscular injections with newly designed cocktail oligos led to high levels of dystrophin expression, with some samples similar to wild-type levels. This is the first report of successful rescue of dystrophin expression with morpholino conjugates in dystrophic dogs. Our results show the potential of phosphorodiamidate morpholino oligomer conjugates as therapeutic agents for DMD. PMID:22888777

  13. Dystrophin-deficient large animal models: translational research and exon skipping

    PubMed Central

    Yu, Xinran; Bao, Bo; Echigoya, Yusuke; Yokota, Toshifumi

    2015-01-01

    Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder caused by mutations in the dystrophin gene. Affecting approximately 1 in 3,600-9337 boys, DMD patients exhibit progressive muscle degeneration leading to fatality as a result of heart or respiratory failure. Despite the severity and prevalence of the disease, there is no cure available. While murine models have been successfully used in illustrating the mechanisms of DMD, their utility in DMD research is limited due to their mild disease phenotypes such as lack of severe skeletal muscle and cardiac symptoms. To address the discrepancy between the severity of disease displayed by murine models and human DMD patients, dystrophin-deficient dog models with a splice site mutation in intron 6 were established. Examples of these are Golden Retriever muscular dystrophy and beagle-based Canine X-linked muscular dystrophy. These large animal models are widely employed in therapeutic DMD research due to their close resemblance to the severity of human patient symptoms. Recently, genetically tailored porcine models of DMD with deleted exon 52 were developed by our group and others, and can potentially act as a new large animal model. While therapeutic outcomes derived from these large animal models can be more reliably extrapolated to DMD patients, a comprehensive understanding of these models is still needed. This paper will discuss recent progress and future directions of DMD studies with large animal models such as canine and porcine models. PMID:26396664

  14. Dystrophin insufficiency causes selective muscle histopathology and loss of dystrophin-glycoprotein complex assembly in pig skeletal muscle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Duchenne muscular dystrophy (DMD) is caused by a dystrophin deficiency while Becker muscular dystrophy (BMD) is caused by a dystrophin insufficiency or expression of a partially functional protein product. Both of these dystrophinopathies are most commonly studied using the mdx mouse and a golden r...

  15. Alternative splicing contributes to K+ channel diversity in the mammalian central nervous system.

    PubMed Central

    Luneau, C J; Williams, J B; Marshall, J; Levitan, E S; Oliva, C; Smith, J S; Antanavage, J; Folander, K; Stein, R B; Swanson, R

    1991-01-01

    In an attempt to define the molecular basis of the functional diversity of K+ channels, we have isolated overlapping rat brain cDNAs that encoded a neuronal delayed rectifier K+ channel, K,4, that is structurally related to the Drosophila Shaw protein. Unlike previously characterized mammalian K+ channel genes, which each contain a single protein-coding exon, K,4 arises from alternative exon usage at a locus that also encodes another mammalian Shaw homolog, NGK2. Thus, the enormous diversity of K+ channels in mammals can be generated not just through gene duplication and divergence but also through alternative splicing of RNA. Images PMID:2023941

  16. Underwater splice for submarine coaxial cable

    SciTech Connect

    Inouye, A.T.; Roe, T. Jr.; Tausing, W.R.; Wilson, J.V.

    1984-10-30

    The invention is a device for splicing submarine coaxial cable underwater on the seafloor with a simple push-on operation to restore and maintain electrical and mechanical strength integrity; the splice device is mateable directly with the severed ends of a coaxial cable to be repaired. Splicing assemblies comprise a dielectric pressure compensating fluid filled guide cavity, a gelled castor oil cap and wiping seals for exclusion of seawater, electrical contacts, a cable strength restoration mechanism, and a pressure compensation system for controlled extrusion of and depletion loss prevention of dielectric seal fluid during cable splicing. A splice is made underwater by directly inserting prepared ends of coaxial cable, having no connector attachments, into splicing assemblies.

  17. Dystrophin Dp71f associates with the beta1-integrin adhesion complex to modulate PC12 cell adhesion

    PubMed Central

    Cerna, Joel; Cerecedo, Doris; Ortega, Arturo; García-Sierra, Francisco; Centeno, Federico; Garrido, Efrain; Mornet, Dominique; Cisneros, Bulmaro

    2006-01-01

    Dystrophin Dp71 is the main product of the Duchenne muscular dystrophy gene in the brain; however, its function is unknown. To study the role of Dp71 in neuronal cells, we previously generated by antisense treatment PC12 neuronal cell clones with decreased Dp71 expression (antisense-Dp71 cells). PC12 cells express two different splicing isoforms of Dp71, a cytoplasmic variant called Dp71f and a nuclear isoform called Dp71d. We previously reported that antisense-Dp71 cells display deficient adhesion to substrate and reduced immunostaining of β1-integrin in the cell area contacting the substrate. In this study, we isolated additional antisenseDp71 clones to analyze in detail the potential involvement of Dp71f isoform with the β1-integrin adhesion system of PC12 cells. Immunofluorescence analyses as well as immunoprecipitation assays demonstrated that the PC12 cell β1-integrin adhesion complex is composed of β1-integrin, talin, paxillin, α-actinin, FAK and actin. In addition, our results showed that Dp71f associates with most of the β1-integrin complex components (β1-integrin, FAK, α-actinin, talin and actin). In the antisense-Dp71 cells, the deficiency of Dp71 provokes a significant reduction of the β1-integrin adhesion complex and, consequently, the deficient adhesion of these cells to laminin. In vitro binding experiments confirmed the interaction of Dp71f with FAK and β1-integrin. Our data indicate that Dp71f is a structural component of the β1-integrin adhesion complex of PC12 cells that modulates PC12 cell adhesion by conferring proper complex assembly and/or maintenance. PMID:16935300

  18. Dystrophin analysis using a panel of anti-dystrophin antibodies in Duchenne and Becker muscular dystrophy.

    PubMed

    Muntoni, F; Mateddu, A; Cianchetti, C; Marrosu, M G; Clerk, A; Cau, M; Congiu, R; Cao, A; Melis, M A

    1993-01-01

    Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, was studied in 19 patients with Xp21 disorders and in 25 individuals with non-Xp21 muscular dystrophy. Antibodies raised to seven different regions spanning most of the protein were used for immunocytochemistry. In all patients specific dystrophin staining anomalies were detected and correlated with clinical severity and also gene deletion. In patients with Becker muscular dystrophy (BMD) the anomalies detected ranged from inter- and intra-fibre variation in labelling intensity with the same antibody or several antibodies to general reduction in staining and discontinuous staining. In vitro evidence of abnormal dystrophin breakdown was observed reanalysing the muscle of patients, with BMD and not that of non-Xp21 dystrophies, after it has been stored for several months. A number of patients with DMD showed some staining but this did not represent a diagnostic problem. Based on the data presented, it was concluded that immunocytochemistry is a powerful technique in the prognostic diagnosis of Xp21 muscular dystrophies. PMID:8429320

  19. Differential expression of dystrophin, utrophin, and dystrophin-associated proteins in human muscle culture.

    PubMed

    Radojevic, V; Lin, S; Burgunder, J M

    2000-06-01

    The dystrophin-associated protein complex (DAP) plays an important role in sarcolemmal function. Mutations of DAP elements lead to diverse forms of muscular dystrophies, among them Duchenne muscular dystrophy, one of the most severe neuromuscular diseases. Strategies in gene therapy are being assessed to restore DAP stability. However, the relationship between DAP elements and time-course of the DAP formation are still not known in detail. In order to better understand the relationship among DAP proteins, we therefore studied their expression during development in human muscle culture in comparison with developmentally regulated muscle proteins. Desmin immunoreactivity (IR) was detected by 3 days in vitro (DIV3), IR for developmental heavy-chain myosin, vimentin, utrophin, and beta-dystroglycan, as well as alpha-, beta-, and gamma-sarcoglycan, a day later. delta-Sarcoglycan was found by DIV7; dystrophin could be detected only by DIV11. In general, DAP proteins were first located in the perinuclear region, later diffusely in the cytoplasm, and finally exclusively at the membrane. This sequence of events during muscle development gives further support to our suggestion that utrophin could be a precursor of dystrophin during development and regeneration. These data also suggest that utrophin alone is sufficient to anchor the complex, which is important when utrophin replacement strategies are being investigated for the treatment of dystrophinopathies. In this study we demonstrated the establishment of a culture technique that should allow the close study of DAP expression in diseased muscle, including its use after gene modulatory strategies. PMID:10928275

  20. Tropomyosin exons as models for alternative splicing.

    PubMed

    Gooding, Clare; Smith, Christopher W J

    2008-01-01

    Three of the four mammalian tropomyosin (Tm) genes are alternatively spliced, most commonly by mutually exclusive selection from pairs of internal or 3' end exons. Alternative splicing events in the TPM1, 2 and 3 genes have been analysed experimentally in various levels ofdetail. In particular, mutually exclusive exon pairs in the betaTm (TPM2) and alphaTm (TPM1) genes are among the most intensively studied models for striated and smooth muscle specific alternative splicing, respectively. Analysis of these model systems has provided important insights into general mechanisms and strategies of splicing regulation. PMID:19209811

  1. Isolated dystrophin molecules as seen by electron microscopy.

    PubMed

    Pons, F; Augier, N; Heilig, R; Léger, J; Mornet, D; Léger, J J

    1990-10-01

    Dystrophin, the protein product of the Duchenne muscular dystrophy locus [Hoffman, E. P., Brown, R. H., Jr., & Kunkel, L. M. (1987) Cell 51, 919-928], is expressed in striated and smooth muscles as well as in non-muscle tissues. Examination of its primary structure has revealed that the molecule is composed of four domains, three of which share many features with the membrane cytoskeletal proteins spectrin and actinin. Dystrophin has thus been predicted to adopt a rod shape [Koenig, M., Monaco, A. P. & Kunkel, L. M. (1988) Cell 53, 219-228]. In the present study, we describe its isolation from the chicken gizzard smooth muscle and present electron microscopic images of the molecule. Polyclonal antibodies were first prepared from a dystrophin fragment derived from the chicken skeletal muscle gene (residues 1173-1728). A dystrophin-enriched membrane preparation from chicken gizzard muscle was then purified by passing it through an affinity chromatography column made with the anti-dystrophin antibodies. Electron microscopy of isolated and rotatory-shadowed dystrophin molecules revealed that the lengths measured for the dystrophin monomers (175 +/- 15 nm) are compatible with a structural arrangement of the repeat sequence segments in triple-barrel alpha-helices connected by short-turn regions, as was earlier postulated for the repeat domains of spectrin and actinin. Electron microscopic images indicate that in addition the dystrophin molecules could present the same capacity of self-association in oligomeric structures as these cytoskeletal proteins and may thus be a part of a complex molecular meshwork essential to muscle cell function. PMID:2236001

  2. RT-PCR analysis of dystrophin mRNA in DND/BMD patients

    SciTech Connect

    Ciafaloni, E.; Silva, H.A.R. de; Roses, A.D.

    1994-09-01

    Duchenne and Becker muscular dystrophies (DMD, BMD) are X-linked recessive disorders caused by mutations in the dystrophin (dys) gene. The majority of these mutations are intragenic deletions of duplications routinely detected by Southern biots and multiplex PCR. The remainder are very likely, smaller mutations, mostly point-mutations. Detection of these mutations is very difficult due to the size and complexity of the dys gene. We applied RT-PCR to analyse the entire dys mRNA of three DMD patients with no detectable genomic defect. In two unrelated patients, a duplication of the 62 bp exon 2 was identified. This causes a frameshift sufficient to explain the DMD phenotype. In the third patient, who had congenital DMD and severe mental retardation, a complex pattern of aberrant splicing at the 3-prime exons 67-79 was observed. Sural nerve biopsy in this patient showed the complete absence of Dp116. PCR-SSCP studies are presently in progress to identify the mutations responsible for the aberrant splicing patterns.

  3. Neural integrity is maintained by dystrophin in C. elegans

    PubMed Central

    Zhou, Shan

    2011-01-01

    The dystrophin protein complex (DPC), composed of dystrophin and associated proteins, is essential for maintaining muscle membrane integrity. The link between mutations in dystrophin and the devastating muscle failure of Duchenne’s muscular dystrophy (DMD) has been well established. Less well appreciated are the accompanying cognitive impairment and neuropsychiatric disorders also presented in many DMD patients, which suggest a wider role for dystrophin in membrane–cytoskeleton function. This study provides genetic evidence of a novel role for DYS-1/dystrophin in maintaining neural organization in Caenorhabditis elegans. This neuronal function is distinct from the established role of DYS-1/dystrophin in maintaining muscle integrity and regulating locomotion. SAX-7, an L1 cell adhesion molecule (CAM) homologue, and STN-2/γ-syntrophin also function to maintain neural integrity in C. elegans. This study provides biochemical data that show that SAX-7 associates with DYS-1 in an STN-2/γ-syntrophin–dependent manner. These results reveal a recruitment of L1CAMs to the DPC to ensure neural integrity is maintained. PMID:21242290

  4. Antisense modulation of both exonic and intronic splicing motifs induces skipping of a DMD pseudo-exon responsible for x-linked dilated cardiomyopathy.

    PubMed

    Rimessi, Paola; Fabris, Marina; Bovolenta, Matteo; Bassi, Elena; Falzarano, Sofia; Gualandi, Francesca; Rapezzi, Claudio; Coccolo, Fabio; Perrone, Daniela; Medici, Alessandro; Ferlini, Alessandra

    2010-09-01

    Antisense-mediated exon skipping has proven to be efficacious for subsets of Duchenne muscular dystrophy mutations. This approach is based on targeting specific splicing motifs that interfere with the spliceosome assembly by steric hindrance. Proper exon recognition by the splicing machinery is thought to depend on exonic splicing enhancer sequences, often characterized by purine-rich stretches, representing potential targets for antisense-mediated exon skipping. We identified and functionally characterized two purine-rich regions located within dystrophin intron 11 and involved in splicing regulation of a pseudo-exon. A functional role for these sequences was suggested by a pure intronic DMD deletion causing X-linked dilated cardiomyopathy through the prevalent cardiac incorporation of the aberrant pseudo-exon, marked as Alu-exon, into the dystrophin transcript. The first splicing sequence is contained within the pseudo-exon, whereas the second is localized within its 3' intron. We demonstrated that the two sequences actually behave as splicing enhancers in cell-free splicing assays because their deletion strongly interferes with the pseudo-exon inclusion. Cell-free results were then confirmed in myogenic cells derived from the patient with X-linked dilated cardiomyopathy, by targeting the identified motifs with antisense molecules and obtaining a reduction in dystrophin pseudo-exon recognition. The splicing motifs identified could represent target sequences for a personalized molecular therapy in this particular DMD mutation. Our results demonstrated for the first time the role of intronic splicing sequences in antisense modulation with implications in exon skipping-mediated therapeutic approaches. PMID:20486769

  5. Gene therapies that restore dystrophin expression for the treatment of Duchenne muscular dystrophy.

    PubMed

    Robinson-Hamm, Jacqueline N; Gersbach, Charles A

    2016-09-01

    Duchenne muscular dystrophy is one of the most common inherited genetic diseases and is caused by mutations to the DMD gene that encodes the dystrophin protein. Recent advances in genome editing and gene therapy offer hope for the development of potential therapeutics. Truncated versions of the DMD gene can be delivered to the affected tissues with viral vectors and show promising results in a variety of animal models. Genome editing with the CRISPR/Cas9 system has recently been used to restore dystrophin expression by deleting one or more exons of the DMD gene in patient cells and in a mouse model that led to functional improvement of muscle strength. Exon skipping with oligonucleotides has been successful in several animal models and evaluated in multiple clinical trials. Next-generation oligonucleotide formulations offer significant promise to build on these results. All these approaches to restoring dystrophin expression are encouraging, but many hurdles remain. This review summarizes the current state of these technologies and summarizes considerations for their future development. PMID:27542949

  6. Gamma1- and gamma2-syntrophins, two novel dystrophin-binding proteins localized in neuronal cells.

    PubMed

    Piluso, G; Mirabella, M; Ricci, E; Belsito, A; Abbondanza, C; Servidei, S; Puca, A A; Tonali, P; Puca, G A; Nigro, V

    2000-05-26

    Dystrophin is the scaffold of a protein complex, disrupted in inherited muscular dystrophies. At the last 3' terminus of the gene, a protein domain is encoded, where syntrophins are tightly bound. These are a family of cytoplasmic peripheral membrane proteins. Three genes have been described encoding one acidic (alpha1) and two basic (beta1 and beta2) proteins of approximately 57-60 kDa. Here, we describe the characterization of two novel putative members of the syntrophin family, named gamma1- and gamma2-syntrophins. The human gamma1-syntrophin gene is composed of 19 exons and encodes a brain-specific protein of 517 amino acids. The human gamma2-syntrophin gene is composed of at least 17 exons, and its transcript is expressed in brain and, to a lesser degree, in other tissues. We mapped the gamma1-syntrophin gene to human chromosome 8q11 and the gamma2-syntrophin gene to chromosome 2p25. Yeast two-hybrid experiments and pull-down studies showed that both proteins can bind the C-terminal region of dystrophin and related proteins. We raised antibodies against these proteins and recognized expression in both rat and human central neurons, coincident with RNA in situ hybridization of adjacent sections. Our present findings suggest a differentiated role of a modified dystrophin-associated complex in the central nervous system. PMID:10747910

  7. Alternative Splice Variants, a New Class of Protein Cancer Biomarker Candidates: Findings in Pancreatic Cancer and Breast Cancer with Systems Biology Implications

    PubMed Central

    Omenn, Gilbert S.; Yocum, Anastasia K.; Menon, Rajasree

    2010-01-01

    Alternative splicing plays an important role in protein diversity without increasing genome size. Earlier thought to be uncommon, splicing appears to affect the majority of genes. Alternative splice variants have been detected at the mRNA level in many diseases. We have designed and demonstrated a discovery pipeline for alternative splice variant (ASV) proteins from tandem MS/MS datasets. We created a modified ECgene database with entries from exhaustive three-frame translation of Ensembl transcripts and gene models from ECgene, with periodic updates. The human database has 14 million entries; the mouse database, 10 million entries. We match MS/MS findings against these potential translation products to identify and quantify known and novel ASVs. In this review, we summarize findings and systems biology implications of biomarker candidates from a mouse model of human pancreatic ductal adenocarcinoma [28] and a mouse model of human Her2/neu-induced breast cancer [27]. The same approach is being applied to human tumors, plasma, and cell line studies of other cancers. PMID:20534909

  8. A Model System for Activation-Induced Alternative Splicing of CD45 Pre-mRNA in T Cells Implicates Protein Kinase C and Ras

    PubMed Central

    Lynch, Kristen W.; Weiss, Arthur

    2000-01-01

    Multiple isoforms of the protein tyrosine phosphatase CD45 are expressed on the surface of human T cells. Interestingly, the expression of these isoforms has been shown to vary significantly upon T-cell activation. In this report, we describe a novel cell line-based model system in which we can mimic the activation-induced alternative splicing of CD45 observed in primary T cells. Of the many proximal signaling events induced by T-cell stimulation, we show that activation of protein kinase C and activation of Ras are important for the switch toward the exclusion of CD45 variable exons, whereas events related to Ca2+ flux are not. In addition, the ability of cycloheximide to block the activation-induced alternative splicing of CD45 suggests a requirement for de novo protein synthesis. We further demonstrate that sequences which have previously been implicated in the tissue-specific regulation of CD45 variable exons are likewise necessary and sufficient for activation-induced splicing. These results provide an initial understanding of the requirements for CD45 alternative splicing upon T-cell activation, and they confirm the importance of this novel cell line in facilitating a more detailed analysis of the activation-induced regulation of CD45 than has been previously possible. PMID:10594010

  9. The lack of the Celf2a splicing factor converts a Duchenne genotype into a Becker phenotype

    PubMed Central

    Martone, J.; Briganti, F.; Legnini, I.; Morlando, M.; Picillo, E.; Sthandier, O.; Politano, L.; Bozzoni, I.

    2016-01-01

    Substitutions, deletions and duplications in the dystrophin gene lead to either the severe Duchenne muscular dystrophy (DMD) or mild Becker muscular dystrophy depending on whether out-of-frame or in-frame transcripts are produced. We identified a DMD case (GSΔ44) where the correlation between genotype and phenotype is not respected, even if carrying a typical Duchenne mutation (exon 44 deletion) a Becker-like phenotype was observed. Here we report that in this patient, partial restoration of an in-frame transcript occurs by natural skipping of exon 45 and that this is due to the lack of Celf2a, a splicing factor that interacts with exon 45 in the dystrophin pre-mRNA. Several experiments are presented that demonstrate the central role of Celf2a in controlling exon 45 splicing; our data point to this factor as a potential target for the improvement of those DMD therapeutic treatments, which requires exon 45 skipping. PMID:26796035

  10. Identification and characterization of a novel retinal isoform of dystrophin

    SciTech Connect

    D`Souza, V.N.; Sigesmund, D.A.; Man, N.

    1994-09-01

    We have shown that dystrophin is required for normal function of the retina as measured by electroretinography (ERG). In these studies a genotype/phenotype correlation was found in which DMD/BMD patients with deletions in the central to distal region of the gene had abnormal ERGs, while patients with deletions in the 5{prime} end of the gene had a mild or normal retinal phenotype. A similar correlation was also observed in the mouse in which the mdx mouse having a mutation in exon 23 had a normal retinal phenotype, whereas the mdx{sup Cv3} mouse (mutation in intron 65) had an abnormal phenotype. Molecular analysis of both human and mouse retina indicated that at least two isoforms of dystrophin are expressed in the retina and localize to the outer plexiform layer, the synaptic junction between the photoreceptors, the bipolar cells, and the horizontal cells. Using a panel of monoclonal dystrophin antisera to analyze mdx mouse retina which does not contain full length dystrophin antisera, we showed that a shorter dystrophin isoform (approximately 260 kDa) was present and contained part of the rod, the cysteine-rich and C-terminal domains. The 5{prime} end of the transcript giving rise to this isoform was characterized and cloned using 5{prime}RACE. Sequence analysis indicated that this transcript contained a novel exon 1 consisting of 240 nucleotides and coded for a unique N-terminus of 13 amino acids. This isoform is distinct from the DP116 dystrophin isoform identified in peripheral nerve. From the functional analysis of DMD patients and dystrophic mice we conclude that this 260 kDa dystrophin isoform is required for normal retinal electrophysiology.

  11. Abnormal splicing switch of DMD's penultimate exon compromises muscle fibre maintenance in myotonic dystrophy.

    PubMed

    Rau, Frédérique; Lainé, Jeanne; Ramanoudjame, Laetitita; Ferry, Arnaud; Arandel, Ludovic; Delalande, Olivier; Jollet, Arnaud; Dingli, Florent; Lee, Kuang-Yung; Peccate, Cécile; Lorain, Stéphanie; Kabashi, Edor; Athanasopoulos, Takis; Koo, Taeyoung; Loew, Damarys; Swanson, Maurice S; Le Rumeur, Elisabeth; Dickson, George; Allamand, Valérie; Marie, Joëlle; Furling, Denis

    2015-01-01

    Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM1. PMID:26018658

  12. Abnormal splicing switch of DMD's penultimate exon compromises muscle fibre maintenance in myotonic dystrophy

    PubMed Central

    Rau, Frédérique; Lainé, Jeanne; Ramanoudjame, Laetitita; Ferry, Arnaud; Arandel, Ludovic; Delalande, Olivier; Jollet, Arnaud; Dingli, Florent; Lee, Kuang-Yung; Peccate, Cécile; Lorain, Stéphanie; Kabashi, Edor; Athanasopoulos, Takis; Koo, Taeyoung; Loew, Damarys; Swanson, Maurice S.; Le Rumeur, Elisabeth; Dickson, George; Allamand, Valérie; Marie, Joëlle; Furling, Denis

    2015-01-01

    Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM1. PMID:26018658

  13. Design and evaluation of locked nucleic acid-based splice-switching oligonucleotides in vitro

    PubMed Central

    Shimo, Takenori; Tachibana, Keisuke; Saito, Kiwamu; Yoshida, Tokuyuki; Tomita, Erisa; Waki, Reiko; Yamamoto, Tsuyoshi; Doi, Takefumi; Inoue, Takao; Kawakami, Junji; Obika, Satoshi

    2014-01-01

    Antisense-mediated modulation of pre-mRNA splicing is an attractive therapeutic strategy for genetic diseases. Currently, there are few examples of modulation of pre-mRNA splicing using locked nucleic acid (LNA) antisense oligonucleotides, and, in particular, no systematic study has addressed the optimal design of LNA-based splice-switching oligonucleotides (LNA SSOs). Here, we designed a series of LNA SSOs complementary to the human dystrophin exon 58 sequence and evaluated their ability to induce exon skipping in vitro using reverse transcription-polymerase chain reaction. We demonstrated that the number of LNAs in the SSO sequence and the melting temperature of the SSOs play important roles in inducing exon skipping and seem to be key factors for designing efficient LNA SSOs. LNA SSO length was an important determinant of activity: a 13-mer with six LNA modifications had the highest efficacy, and a 7-mer was the minimal length required to induce exon skipping. Evaluation of exon skipping activity using mismatched LNA/DNA mixmers revealed that 9-mer LNA SSO allowed a better mismatch discrimination. LNA SSOs also induced exon skipping of endogenous human dystrophin in primary human skeletal muscle cells. Taken together, our findings indicate that LNA SSOs are powerful tools for modulating pre-mRNA splicing. PMID:24935206

  14. The evolution of spliced leader trans-splicing in nematodes.

    PubMed

    Pettitt, Jonathan; Harrison, Neale; Stansfield, Ian; Connolly, Bernadette; Müller, Berndt

    2010-08-01

    Spliced leader trans-splicing occurs in many primitive eukaryotes including nematodes. Most of our knowledge of trans-splicing in nematodes stems from the model organism Caenorhabditis elegans and relatives, and from work with Ascaris. Our investigation of spliced leader trans-splicing in distantly related Dorylaimia nematodes indicates that spliced-leader trans-splicing arose before the nematode phylum and suggests that the spliced leader RNA gene complements in extant nematodes have evolved from a common ancestor with a diverse set of spliced leader RNA genes. PMID:20659016

  15. Dystrophin insufficiency causes a Becker muscular dystrophy-like phenotype in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Duchenne muscular dystrophy (DMD) is caused by a dystrophin deficiency while Becker MD is caused by a dystrophin insufficiency or expression of a partially functional dystrophin protein. Deficiencies in existing mouse and dog models necessitate the development of a novel large animal model. Our pu...

  16. Metabolic and Signaling Alterations in Dystrophin-Deficient Hearts Precede Overt Cardiomyopathy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cytoskeletal protein dystrophin has been implicated in hereditary and acquired forms of cardiomyopathy. However, much remains to be learned about the role of dystrophin in the heart. We hypothesized that the dystrophin-deficient heart displays early alterations in energy metabolism that precede ...

  17. Adipocyte Spliced Form of X-Box–Binding Protein 1 Promotes Adiponectin Multimerization and Systemic Glucose Homeostasis

    PubMed Central

    Sha, Haibo; Yang, Liu; Liu, Meilian; Xia, Sheng; Liu, Yong; Liu, Feng; Kersten, Sander; Qi, Ling

    2014-01-01

    The physiological role of the spliced form of X-box–binding protein 1 (XBP1s), a key transcription factor of the endoplasmic reticulum (ER) stress response, in adipose tissue remains largely unknown. In this study, we show that overexpression of XBP1s promotes adiponectin multimerization in adipocytes, thereby regulating systemic glucose homeostasis. Ectopic expression of XBP1s in adipocytes improves glucose tolerance and insulin sensitivity in both lean and obese (ob/ob) mice. The beneficial effect of adipocyte XBP1s on glucose homeostasis is associated with elevated serum levels of high-molecular-weight adiponectin and, indeed, is adiponectin-dependent. Mechanistically, XBP1s promotes adiponectin multimerization rather than activating its transcription, likely through a direct regulation of the expression of several ER chaperones involved in adiponectin maturation, including glucose-regulated protein 78 kDa, protein disulfide isomerase family A, member 6, ER protein 44, and disulfide bond oxidoreductase A–like protein. Thus, we conclude that XBP1s is an important regulator of adiponectin multimerization, which may lead to a new therapeutic approach for the treatment of type 2 diabetes and hypoadiponectinemia. PMID:24241534

  18. Adipocyte spliced form of X-box-binding protein 1 promotes adiponectin multimerization and systemic glucose homeostasis.

    PubMed

    Sha, Haibo; Yang, Liu; Liu, Meilian; Xia, Sheng; Liu, Yong; Liu, Feng; Kersten, Sander; Qi, Ling

    2014-03-01

    The physiological role of the spliced form of X-box-binding protein 1 (XBP1s), a key transcription factor of the endoplasmic reticulum (ER) stress response, in adipose tissue remains largely unknown. In this study, we show that overexpression of XBP1s promotes adiponectin multimerization in adipocytes, thereby regulating systemic glucose homeostasis. Ectopic expression of XBP1s in adipocytes improves glucose tolerance and insulin sensitivity in both lean and obese (ob/ob) mice. The beneficial effect of adipocyte XBP1s on glucose homeostasis is associated with elevated serum levels of high-molecular-weight adiponectin and, indeed, is adiponectin-dependent. Mechanistically, XBP1s promotes adiponectin multimerization rather than activating its transcription, likely through a direct regulation of the expression of several ER chaperones involved in adiponectin maturation, including glucose-regulated protein 78 kDa, protein disulfide isomerase family A, member 6, ER protein 44, and disulfide bond oxidoreductase A-like protein. Thus, we conclude that XBP1s is an important regulator of adiponectin multimerization, which may lead to a new therapeutic approach for the treatment of type 2 diabetes and hypoadiponectinemia. PMID:24241534

  19. Read-through compound 13 restores dystrophin expression and improves muscle function in the mdx mouse model for Duchenne muscular dystrophy

    PubMed Central

    Kayali, Refik; Ku, Jin-Mo; Khitrov, Gregory; Jung, Michael E.; Prikhodko, Olga; Bertoni, Carmen

    2012-01-01

    Molecules that induce ribosomal read-through of nonsense mutations in mRNA and allow production of a full-length functional protein hold great therapeutic potential for the treatment of many genetic disorders. Two such read-through compounds, RTC13 and RTC14, were recently identified by a luciferase-independent high-throughput screening assay and were shown to have potential therapeutic functions in the treatment of nonsense mutations in the ATM and the dystrophin genes. We have now tested the ability of RTC13 and RTC14 to restore dystrophin expression into skeletal muscles of the mdx mouse model for Duchenne muscular dystrophy (DMD). Direct intramuscular injection of compound RTC14 did not result in significant read-through activity in vivo and demonstrated the levels of dystrophin protein similar to those detected using gentamicin. In contrast, significant higher amounts of dystrophin were detected after intramuscular injection of RTC13. When administered systemically, RTC13 was shown to partially restore dystrophin protein in different muscle groups, including diaphragm and heart, and improved muscle function. An increase in muscle strength was detected in all treated animals and was accompanied by a significant decrease in creatine kinase levels. These studies establish the therapeutic potential of RTC13 in vivo and advance this newly identified compound into preclinical application for DMD. PMID:22692682

  20. Read-through compound 13 restores dystrophin expression and improves muscle function in the mdx mouse model for Duchenne muscular dystrophy.

    PubMed

    Kayali, Refik; Ku, Jin-Mo; Khitrov, Gregory; Jung, Michael E; Prikhodko, Olga; Bertoni, Carmen

    2012-09-15

    Molecules that induce ribosomal read-through of nonsense mutations in mRNA and allow production of a full-length functional protein hold great therapeutic potential for the treatment of many genetic disorders. Two such read-through compounds, RTC13 and RTC14, were recently identified by a luciferase-independent high-throughput screening assay and were shown to have potential therapeutic functions in the treatment of nonsense mutations in the ATM and the dystrophin genes. We have now tested the ability of RTC13 and RTC14 to restore dystrophin expression into skeletal muscles of the mdx mouse model for Duchenne muscular dystrophy (DMD). Direct intramuscular injection of compound RTC14 did not result in significant read-through activity in vivo and demonstrated the levels of dystrophin protein similar to those detected using gentamicin. In contrast, significant higher amounts of dystrophin were detected after intramuscular injection of RTC13. When administered systemically, RTC13 was shown to partially restore dystrophin protein in different muscle groups, including diaphragm and heart, and improved muscle function. An increase in muscle strength was detected in all treated animals and was accompanied by a significant decrease in creatine kinase levels. These studies establish the therapeutic potential of RTC13 in vivo and advance this newly identified compound into preclinical application for DMD. PMID:22692682

  1. Alternative Splicing in CKD.

    PubMed

    Stevens, Megan; Oltean, Sebastian

    2016-06-01

    Alternative splicing (AS) has emerged in the postgenomic era as one of the main drivers of proteome diversity, with ≥94% of multiexon genes alternatively spliced in humans. AS is therefore one of the main control mechanisms for cell phenotype, and is a process deregulated in disease. Numerous reports describe pathogenic mutations in splice factors, splice sites, or regulatory sequences. Additionally, compared with the physiologic state, disease often associates with an abnormal proportion of splice isoforms (or novel isoforms), without an apparent driver mutation. It is therefore essential to study how AS is regulated in physiology, how it contributes to pathogenesis, and whether we can manipulate faulty splicing for therapeutic advantage. Although the disease most commonly linked to deregulation of AS in several genes is cancer, many reports detail pathogenic splice variants in diseases ranging from neuromuscular disorders to diabetes or cardiomyopathies. A plethora of splice variants have been implicated in CKDs as well. In this review, we describe examples of these CKD-associated splice variants and ideas on how to manipulate them for therapeutic benefit. PMID:26763787

  2. Immobility reduces muscle fiber necrosis in dystrophin deficient muscular dystrophy.

    PubMed

    Kimura, S; Ikezawa, M; Nomura, K; Ito, K; Ozasa, S; Ueno, H; Yoshioka, K; Yano, S; Yamashita, T; Matuskura, M; Miike, T

    2006-08-01

    Duchenne/Becker muscular dystrophy is a progressive muscle disease, which is caused by the abnormality of dystrophin. Spina bifida is characterized by paralysis of the feet, with most of the upper extremities not being affected. We report here on the first case of Becker muscular dystrophy coinciding with spina bifida. The muscle biopsy specimens of the patient showed dystrophic changes in upper extremities, but clearly less in lower extremities. The results show that the restriction of excessive exercise is important for dystrophin deficiency disease. PMID:16516424

  3. Arabidopsis PTB1 and PTB2 proteins negatively regulate splicing of a mini-exon splicing reporter and affect alternative splicing of endogenous genes differentially.

    PubMed

    Simpson, Craig G; Lewandowska, Dominika; Liney, Michele; Davidson, Diane; Chapman, Sean; Fuller, John; McNicol, Jim; Shaw, Paul; Brown, John W S

    2014-07-01

    This paper examines the function of Arabidopsis thaliana AtPTB1 and AtPTB2 as plant splicing factors. The effect on splicing of overexpression of AtPTB1 and AtPTB2 was analysed in an in vivo protoplast transient expression system with a novel mini-exon splicing reporter. A range of mutations in pyrimidine-rich sequences were compared with and without AtPTB and NpU2AF65 overexpression. Splicing analyses of constructs in protoplasts and RNA from overexpression lines used high-resolution reverse transcription polymerase chain reaction (RT-PCR). AtPTB1 and AtPTB2 reduced inclusion/splicing of the potato invertase mini-exon splicing reporter, indicating that these proteins can repress plant intron splicing. Mutation of the polypyrimidine tract and closely associated Cytosine and Uracil-rich (CU-rich) sequences, upstream of the mini-exon, altered repression by AtPTB1 and AtPTB2. Coexpression of a plant orthologue of U2AF65 alleviated the splicing repression of AtPTB1. Mutation of a second CU-rich upstream of the mini-exon 3' splice site led to a decline in mini-exon splicing, indicating the presence of a splicing enhancer sequence. Finally, RT-PCR of AtPTB overexpression lines with c. 90 known alternative splicing (AS) events showed that AtPTBs significantly altered AS of over half the events. AtPTB1 and AtPTB2 are splicing factors that influence alternative splicing. This occurs in the potato invertase mini-exon via the polypyrimidine tract and associated pyrimidine-rich sequence. PMID:24749484

  4. A Duchenne Muscular Dystrophy Gene Hot Spot Mutation in Dystrophin-Deficient Cavalier King Charles Spaniels Is Amenable to Exon 51 Skipping

    PubMed Central

    Walmsley, Gemma L.; Arechavala-Gomeza, Virginia; Fernandez-Fuente, Marta; Burke, Margaret M.; Nagel, Nicole; Holder, Angela; Stanley, Rachael; Chandler, Kate; Marks, Stanley L.; Muntoni, Francesco; Shelton, G. Diane; Piercy, Richard J.

    2010-01-01

    Background Duchenne muscular dystrophy (DMD), which afflicts 1 in 3500 boys, is one of the most common genetic disorders of children. This fatal degenerative condition is caused by an absence or deficiency of dystrophin in striated muscle. Most affected patients have inherited or spontaneous deletions in the dystrophin gene that disrupt the reading frame resulting in unstable truncated products. For these patients, restoration of the reading frame via antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach. The major DMD deletion “hot spot” is found between exons 45 and 53, and skipping exon 51 in particular is predicted to ameliorate the dystrophic phenotype in the greatest number of patients. Currently the mdx mouse is the most widely used animal model of DMD, although its mild phenotype limits its suitability in clinical trials. The Golden Retriever muscular dystrophy (GRMD) model has a severe phenotype, but due to its large size, is expensive to use. Both these models have mutations in regions of the dystrophin gene distant from the commonly mutated DMD “hot spot”. Methodology/Principal Findings Here we describe the severe phenotype, histopathological findings, and molecular analysis of Cavalier King Charles Spaniels with dystrophin-deficient muscular dystrophy (CKCS-MD). The dogs harbour a missense mutation in the 5′ donor splice site of exon 50 that results in deletion of exon 50 in mRNA transcripts and a predicted premature truncation of the translated protein. Antisense oligonucleotide-mediated skipping of exon 51 in cultured myoblasts from an affected dog restored the reading frame and protein expression. Conclusions/Significance Given the small size of the breed, the amiable temperament and the nature of the mutation, we propose that CKCS-MD is a valuable new model for clinical trials of antisense oligonucleotide-induced exon skipping and other therapeutic approaches for DMD. PMID:20072625

  5. Dystrophin is a tumor suppressor in human cancers with myogenic programs.

    PubMed

    Wang, Yuexiang; Marino-Enriquez, Adrian; Bennett, Richard R; Zhu, Meijun; Shen, Yiping; Eilers, Grant; Lee, Jen-Chieh; Henze, Joern; Fletcher, Benjamin S; Gu, Zhizhan; Fox, Edward A; Antonescu, Cristina R; Fletcher, Christopher D M; Guo, Xiangqian; Raut, Chandrajit P; Demetri, George D; van de Rijn, Matt; Ordog, Tamas; Kunkel, Louis M; Fletcher, Jonathan A

    2014-06-01

    Many common human mesenchymal tumors, including gastrointestinal stromal tumor (GIST), rhabdomyosarcoma (RMS) and leiomyosarcoma (LMS), feature myogenic differentiation. Here we report that intragenic deletion of the dystrophin-encoding and muscular dystrophy-associated DMD gene is a frequent mechanism by which myogenic tumors progress to high-grade, lethal sarcomas. Dystrophin is expressed in the non-neoplastic and benign counterparts of GIST, RMS and LMS tumors, and DMD deletions inactivate larger dystrophin isoforms, including 427-kDa dystrophin, while preserving the expression of an essential 71-kDa isoform. Dystrophin inhibits myogenic sarcoma cell migration, invasion, anchorage independence and invadopodia formation, and dystrophin inactivation was found in 96%, 100% and 62% of metastatic GIST, embryonal RMS and LMS samples, respectively. These findings validate dystrophin as a tumor suppressor and likely anti-metastatic factor, suggesting that therapies in development for muscular dystrophies may also have relevance in the treatment of cancer. PMID:24793134

  6. Dystrophin, utrophin and {beta}-dystroglycan expression in skeletal muscle from patients with Becker muscular dystrophy

    SciTech Connect

    Kawajiri, Masakazu; Mitsui, Takao; Kawai, Hisaomi

    1996-08-01

    The precise localization and semiquantitative correlation of dystrophin, utrophin and {beta}-dystroglycan expression on the sarcolemma of skeletal muscle cells obtained from patients with Becker muscular dystrophy (BMD) was studied using three types of double immunofluorescence. Staining intensity was measured using a confocal laser microscope. Each of these proteins was identified at the same locus on the sarcolemma. The staining intensities of dystrophin and utrophin were approximately reciprocal at sarcolemmal sites where dystrophin expression was obviously observed. The staining intensity of {beta}-dystroglycan was strong in areas where dystrophin staining was also strong and utrophin expression was weak. Quantitative analysis revealed that the staining intensity of {beta}-dystroglycan minus that of dystrophin approximated the staining intensity of utrophin, indicating that the sum of dystrophin and utrophin expression corresponds to that of {beta}-dystroglycan. These results suggest that utrophin may compensate for dystrophin deficiency found in BMD by binding to {beta}-dystroglycan. 35 refs., 3 figs., 1 tab.

  7. A PCR-based assay for the wild-type dystrophin gene transferred into the mdx mouse.

    PubMed

    Shrager, J B; Naji, A; Kelly, A M; Stedman, H H

    1992-10-01

    Myoblast transfer has emerged as a promising treatment for inherited myopathies such as Duchenne muscular dystrophy (DMD). Further development of the technique's therapeutic potential requires an experimental system in which issues of graft rejection can be clearly discriminated from those related to myoblast biology. Here we report the development and initial application of a quantitative assay for myogenic cells bearing a wild-type dystrophin gene following transfer into the mdx mouse. The technique relies upon the ability of a mutagenizing polymerase chain reaction (PCR) primer to create a new restriction site in the amplification production of the wild-type, but not the mdx dystrophin gene. The ratio of host to donor cells can be determined from muscle biopsies as small as 1 mg, regardless of donor H-2 background. This simple technique should allow a number of basic questions related to myoblast and direct gene transfer to be addressed using the mdx mouse model. PMID:1357549

  8. Electrical-splicing connector

    NASA Technical Reports Server (NTRS)

    Stringer, E. J.

    1977-01-01

    Connection can be made without removing insulation, and connector case insulates splice. Device can be made in various sizes and saves time, especially when working on prototype boards with several interconnecting test leads.

  9. Monoclonal antibody evidence for structural similarities between the central rod regions of actinin and dystrophin.

    PubMed

    Nguyen, T M; Ellis, J M; Ginjaar, I B; van Paassen, M M; van Ommen, G J; Moorman, A F; Cartwright, A J; Morris, G E

    1990-10-15

    A monoclonal antibody, MANDYS141, binds to both dystrophin and actinin on Western blots (SDS-denatured), but only to actinin in frozen sections of human muscle (native conformation). It differs from a polyclonal cross-reacting antiserum in that it binds to several muscle isoforms of actinin (smooth, fast and slow) from man, mouse and chicken and recognises a quite different part of the proposed triple-helical region of dystrophin (amino acids 1750-2248). The results suggest that structural homologies between actinin and dystrophin occur more than once in their central helical regions and provide experimental support for an actinin-like central rod model for dystrophin. PMID:1699800

  10. Evolutionary study of vertebrate and invertebrate members of the dystrophin and utrophin gene family

    SciTech Connect

    Roberts, R.G.; Nicholson, L.; Bobrow, M.

    1994-09-01

    Vertebrates express two members of the dystrophin gene family. The prototype, dystrophin, is expressed in muscle and neural tissue, and is defective in the human disorders Duchenne and Becker muscular dystrophy (DMD, BMD). The dystrophin homologue utrophin is more generally expressed but has not yet been associated with a genetic disorder. The function of neither protein is clear. A comparison of human utrophin with the known dystrophins (human, mouse, chicken, Torpedo) suggests that dystrophin and utrophin diverged before the vertebrate radiation. We have used reverse-transcript PCR (RT-PCR) directed by degenerate primers to characterize dystrophin and utrophin transcripts from a range of vertebrate and invertebrate animals. Our results suggest that the duplication leading to distinct dystrophin and utrophin genes occurred close to the point of divergence of urochordates from the cephalochordate-vertebrate lineage. This divergence may have occurred to fulfill a novel role which arose at this point, or may reflect a need for separate regulation of the neuromuscular and other functions of the ancient dystrophin. Our data include sequences of the first non-human utrophins to be characterized, and show these to be substantially more divergent than their cognate dystrophins. In addition, our results provide a large body of information regarding the tolerance of amino acid positions in the cysteine-rich and C-terminal domains to substitution. This will aid the interpretations of DMD and BMD missense mutations in these regions.

  11. Analysis of differential splicing suggests different modes of short-term splicing regulation

    PubMed Central

    Topa, Hande; Honkela, Antti

    2016-01-01

    Motivation: Alternative splicing is an important mechanism in which the regions of pre-mRNAs are differentially joined in order to form different transcript isoforms. Alternative splicing is involved in the regulation of normal physiological functions but also linked to the development of diseases such as cancer. We analyse differential expression and splicing using RNA-sequencing time series in three different settings: overall gene expression levels, absolute transcript expression levels and relative transcript expression levels. Results: Using estrogen receptor α signaling response as a model system, our Gaussian process-based test identifies genes with differential splicing and/or differentially expressed transcripts. We discover genes with consistent changes in alternative splicing independent of changes in absolute expression and genes where some transcripts change whereas others stay constant in absolute level. The results suggest classes of genes with different modes of alternative splicing regulation during the experiment. Availability and Implementation: R and Matlab codes implementing the method are available at https://github.com/PROBIC/diffsplicing. An interactive browser for viewing all model fits is available at http://users.ics.aalto.fi/hande/splicingGP/ Contact: hande.topa@helsinki.fi or antti.honkela@helsinki.fi Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27307611

  12. piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts

    PubMed Central

    Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K.

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. PMID:26682797

  13. piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts.

    PubMed

    Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K

    2016-01-29

    Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. PMID:26682797

  14. An ENU Mutagenesis Screen in Zebrafish for Visual System Mutants Identifies a Novel Splice-Acceptor Site Mutation in patched2 that Results in Colobomas

    PubMed Central

    Lee, Jiwoon; Cox, Ben D.; Daly, Christina M. S.; Lee, Chanjae; Nuckels, Richard J.; Tittle, Rachel K.; Uribe, Rosa A.; Gross, Jeffrey M.

    2012-01-01

    Purpose. To identify recessive mutations affecting development and/or maintenance of the zebrafish visual system. Methods. A three-generation ENU (N-Nitroso-N-ethylurea)-based forward genetic screen was performed. F3 embryos were screened visually from 1 to 5 days postfertilization (dpf) for ocular abnormalities, and 5 dpf embryos were fixed and processed for cryosectioning, after which eye sections were screened for defects in cellular organization within the retina, lens, and cornea. A combination of PCR and DNA sequencing, in situ hybridization, and pharmacological treatments were used to clone and characterize a coloboma mutant. Results. A total of 126 F2 families were screened, and, from these, 18 recessive mutations were identified that affected eye development. Phenotypes included lens malformations and cataracts, photoreceptor defects, oculocutaneous albinism, microphthalmia, and colobomas. Analysis of one such coloboma mutant, uta1, identified a splice-acceptor mutation in the patched2 gene that resulted in an in-frame deletion of 19 amino acids that are predicted to contribute to the first extracellular loop of Patched2. ptch2uta1 mutants possessed elevated Hedgehog (Hh) pathway activity, and blocking the Hh pathway with cyclopamine prevented colobomas in ptch2uta1 mutant embryos. Conclusions. We have identified 18 recessive mutations affecting development of the zebrafish visual system and we have characterized a novel splice-acceptor site mutation in patched2 that results in enhanced Hh pathway activity and colobomas. PMID:23150614

  15. Spectrum of small mutations in the dystrophin coding region

    SciTech Connect

    Prior, T.W.; Bartolo, C.; Pearl, D.K.

    1995-07-01

    Duchenne and Becker muscular dystrophies (DMD and BMD) are caused by defects in the dystrophin gene. About two-thirds of the affected patients have large deletions or duplications, which occur in the 5` and central portion of the gene. The nondeletion/duplication cases are most likely the result of smaller mutations that cannot be identified by current diagnostic screening strategies. We screened {approximately} 80% of the dystrophin coding sequence for small mutations in 158 patients without deletions or duplications and identified 29 mutations. The study indicates that many of the DMD and the majority of the BMD small mutations lie in noncoding regions of the gene. All of the mutations identified were unique to single patients, and most of the mutations resulted in protein truncation. We did not find a clustering of small mutations similar to the deletion distribution but found > 40% of the small mutations 3` of exon 55. The extent of protein truncation caused by the 3` mutations did not determine the phenotype, since even the exon 76 nonsense mutation resulted in the severe DMD phenotype. Our study confirms that the dystrophin gene is subject to a high rate of mutation in CpG sequences. As a consequence of not finding any hotspots or prevalent small mutations, we conclude that it is presently not possible to perform direct carrier and prenatal diagnostics for many families without deletions or duplications. 71 refs., 2 figs., 2 tabs.

  16. In Vivo Fusion of Circulating Fluorescent Cells with Dystrophin-Deficient Myofibers Results in Extensive Sarcoplasmic Fluorescence Expression but Limited Dystrophin Sarcolemmal Expression

    PubMed Central

    Chretien, Fabrice; Dreyfus, Patrick A.; Christov, Christo; Caramelle, Philippe; Lagrange, Jean-Léon; Chazaud, Bénédicte; Gherardi, Romain K.

    2005-01-01

    To investigate the therapeutic potential of bone marrow transplantation in Duchenne muscular dystrophy, green fluorescent protein-positive (GFP+) bone marrow cells were transplanted into irradiated wild-type and dystrophin-deficient mdx mice. Tibialis anterior muscles showed fivefold to sixfold more GFP+ mononucleated cells and threefold to fourfold more GFP+ myofibers in mdx than in wild-type mice. In contrast, dystrophin expression in mdx mice remained within the level of nontransplanted mdx mice, and co-expression with GFP was rare. Longitudinal sections of 5000 myofibers showed 160 GFP+ fibers, including 9 that co-expressed dystrophin. GFP was always visualized as full-length sarcoplasmic fluorescence that exceeded the span of sample length (up to 1500 μm), whereas dystrophin expression was restricted to 11 to 28% of this length. Dystrophin expression span was much shorter in GFP+ fibers (116 ± 46 μm) than in revertant fibers (654 ± 409 μm). These data suggest that soluble GFP diffuses far from the fusion site with a pre-existing dystrophin− myofiber whereas dystrophin remains mainly expressed close to the site of fusion. Because restoration of dystrophin in whole muscle fiber length is required to expect functional improvement and clinical benefits for Duchenne muscular dystrophy, future applications of cell therapies to neuromuscular disorders could be more appropriately envisaged for replacement of defective soluble sarcoplasmic proteins. PMID:15920159

  17. Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process

    PubMed Central

    Meyer, Katja; Koester, Tino; Staiger, Dorothee

    2015-01-01

    Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance. PMID:26213982

  18. Evolution of alternative splicing regulation: changes in predicted exonic splicing regulators are not associated with changes in alternative splicing levels in primates.

    PubMed

    Irimia, Manuel; Rukov, Jakob Lewin; Roy, Scott William

    2009-01-01

    Alternative splicing is tightly regulated in a spatio-temporal and quantitative manner. This regulation is achieved by a complex interplay between spliceosomal (trans) factors that bind to different sequence (cis) elements. cis-elements reside in both introns and exons and may either enhance or silence splicing. Differential combinations of cis-elements allows for a huge diversity of overall splicing signals, together comprising a complex 'splicing code'. Many cis-elements have been identified, and their effects on exon inclusion levels demonstrated in reporter systems. However, the impact of interspecific differences in these elements on the evolution of alternative splicing levels has not yet been investigated at genomic level. Here we study the effect of interspecific differences in predicted exonic splicing regulators (ESRs) on exon inclusion levels in human and chimpanzee. For this purpose, we compiled and studied comprehensive datasets of predicted ESRs, identified by several computational and experimental approaches, as well as microarray data for changes in alternative splicing levels between human and chimpanzee. Surprisingly, we found no association between changes in predicted ESRs and changes in alternative splicing levels. This observation holds across different ESR exon positions, exon lengths, and 5' splice site strengths. We suggest that this lack of association is mainly due to the great importance of context for ESR functionality: many ESR-like motifs in primates may have little or no effect on splicing, and thus interspecific changes at short-time scales may primarily occur in these effectively neutral ESRs. These results underscore the difficulties of using current computational ESR prediction algorithms to identify truly functionally important motifs, and provide a cautionary tale for studies of the effect of SNPs on splicing in human disease. PMID:19495418

  19. Clinical and molecular characterization of a cohort of patients with novel nucleotide alterations of the Dystrophin gene detected by direct sequencing

    PubMed Central

    2011-01-01

    Background Duchenne and Becker Muscular dystrophies (DMD/BMD) are allelic disorders caused by mutations in the dystrophin gene, which encodes a sarcolemmal protein responsible for muscle integrity. Deletions and duplications account for approximately 75% of mutations in DMD and 85% in BMD. The implementation of techniques allowing complete gene sequencing has focused attention on small point mutations and other mechanisms underlying complex rearrangements. Methods We selected 47 patients (41 families; 35 DMD, 6 BMD) without deletions and duplications in DMD gene (excluded by multiplex ligation-dependent probe amplification and multiplex polymerase chain reaction analysis). This cohort was investigated by systematic direct sequence analysis to study sequence variation. We focused our attention on rare mutational events which were further studied through transcript analysis. Results We identified 40 different nucleotide alterations in DMD gene and their clinical correlates; altogether, 16 mutations were novel. DMD probands carried 9 microinsertions/microdeletions, 19 nonsense mutations, and 7 splice-site mutations. BMD patients carried 2 nonsense mutations, 2 splice-site mutations, 1 missense substitution, and 1 single base insertion. The most frequent stop codon was TGA (n = 10 patients), followed by TAG (n = 7) and TAA (n = 4). We also analyzed the molecular mechanisms of five rare mutational events. They are two frame-shifting mutations in the DMD gene 3'end in BMD and three novel splicing defects: IVS42: c.6118-3C>A, which causes a leaky splice-site; c.9560A>G, which determines a cryptic splice-site activation and c.9564-426 T>G, which creates pseudoexon retention within IVS65. Conclusion The analysis of our patients' sample, carrying point mutations or complex rearrangements in DMD gene, contributes to the knowledge on phenotypic correlations in dystrophinopatic patients and can provide a better understanding of pre-mRNA maturation defects and dystrophin

  20. Viral interactions with components of the splicing machinery.

    PubMed

    Meyer, F

    2016-01-01

    Eukaryotic genes are often interrupted by stretches of sequence with no protein coding potential or obvious function. After transcription, these interrupting sequences must be removed to give rise to the mature messenger RNA. This fundamental process is called RNA splicing and is achieved by complicated machinery made of protein and RNA that assembles around the RNA to be edited. Viruses also use RNA splicing to maximize their coding potential and economize on genetic space, and use clever strategies to manipulate the splicing machinery to their advantage. This article gives an overview of the splicing process and provides examples of viral strategies that make use of various components of the splicing system to promote their replicative cycle. Representative virus families have been selected to illustrate the interaction with various regulatory proteins and ribonucleoproteins. The unifying theme is fine regulation through protein-protein and protein-RNA interactions with the spliceosome components and associated factors to promote or prevent spliceosome assembly on given splice sites, in addition to a strong influence from cis-regulatory sequences on viral transcripts. Because there is an intimate coupling of splicing with the processes that direct mRNA biogenesis, a description of how these viruses couple the regulation of splicing with the retention or stability of mRNAs is also included. It seems that a unique balance of suppression and activation of splicing and nuclear export works optimally for each family of viruses. PMID:27571697

  1. Spliced leader trans-splicing in the nematode Trichinella spiralis uses highly polymorphic, noncanonical spliced leaders.

    PubMed

    Pettitt, Jonathan; Müller, Berndt; Stansfield, Ian; Connolly, Bernadette

    2008-04-01

    The trans-splicing of short spliced leader (SL) RNAs onto the 5' ends of mRNAs occurs in a diverse range of taxa. In nematodes, all species so far characterized utilize a characteristic, conserved spliced leader, SL1, as well as variants that are employed in the resolution of operons. Here we report the identification of spliced leader trans-splicing in the basal nematode Trichinella spiralis, and show that this nematode does not possess a canonical SL1, but rather has at least 15 distinct spliced leaders, encoded by at least 19 SL RNA genes. The individual spliced leaders vary in both size and primary sequence, showing a much higher degree of diversity compared to other known trans-spliced leaders. In a survey of T. spiralis mRNAs, individual mRNAs were found to be trans-spliced to a number of different spliced leader sequences. These data provide the first indication that the last common ancestor of the phylum Nematoda utilized spliced leader trans-splicing and that the canonical spliced leader, SL1, found in Caenorhabditis elegans, evolved after the divergence of the major nematode clades. This discovery sheds important light on the nature and evolution of mRNA processing in the Nematoda. PMID:18256244

  2. Variation in alternative splicing across human tissues

    PubMed Central

    Yeo, Gene; Holste, Dirk; Kreiman, Gabriel; Burge, Christopher B

    2004-01-01

    Background Alternative pre-mRNA splicing (AS) is widely used by higher eukaryotes to generate different protein isoforms in specific cell or tissue types. To compare AS events across human tissues, we analyzed the splicing patterns of genomically aligned expressed sequence tags (ESTs) derived from libraries of cDNAs from different tissues. Results Controlling for differences in EST coverage among tissues, we found that the brain and testis had the highest levels of exon skipping. The most pronounced differences between tissues were seen for the frequencies of alternative 3' splice site and alternative 5' splice site usage, which were about 50 to 100% higher in the liver than in any other human tissue studied. Quantifying differences in splice junction usage, the brain, pancreas, liver and the peripheral nervous system had the most distinctive patterns of AS. Analysis of available microarray expression data showed that the liver had the most divergent pattern of expression of serine-arginine protein and heterogeneous ribonucleoprotein genes compared to the other human tissues studied, possibly contributing to the unusually high frequency of alternative splice site usage seen in liver. Sequence motifs enriched in alternative exons in genes expressed in the brain, testis and liver suggest specific splicing factors that may be important in AS regulation in these tissues. Conclusions This study distinguishes the human brain, testis and liver as having unusually high levels of AS, highlights differences in the types of AS occurring commonly in different tissues, and identifies candidate cis-regulatory elements and trans-acting factors likely to have important roles in tissue-specific AS in human cells. PMID:15461793

  3. Expression of recombinant dystrophin and its localization to the cell membrane.

    PubMed

    Lee, C C; Pearlman, J A; Chamberlain, J S; Caskey, C T

    1991-01-24

    Duchenne's muscular dystrophy (DMD) is an X-linked progressive myopathy caused by a defect in the DMD gene locus. The gene corresponding to the DMD locus produces a 14-kilobase (kb) messenger RNA that codes for a large cytoskeletal membrane protein, dystrophin. DMD and Becker's muscular dystrophy are the consequences of dystrophin mutations. The exact biological function of dystrophin remains unknown but it has been demonstrated that it is localized to the cytoplasmic face of the cell membrane and has direct interaction with several other membrane proteins. We report here the synthesis of a 14-kb full-length complementary DNA for the mouse muscle dystrophin mRNA and the expression of this cDNA in COS cells. The recombinant dystrophin is indistinguishable from mouse muscle dystrophin by western blot analysis with anti-dystrophin antibodies and was shown by an immunofluorescent technique to be localized in the cell membrane. Our successful construction of a functional full-length cDNA opens opportunities for the study of structure and function of dystrophin and provides an opportunity to initiate gene therapy studies. PMID:1824797

  4. In vivo dynamics of skeletal muscle Dystrophin in zebrafish embryos revealed by improved FRAP analysis

    PubMed Central

    Bajanca, Fernanda; Gonzalez-Perez, Vinicio; Gillespie, Sean J; Beley, Cyriaque; Garcia, Luis; Theveneau, Eric; Sear, Richard P; Hughes, Simon M

    2015-01-01

    Dystrophin forms an essential link between sarcolemma and cytoskeleton, perturbation of which causes muscular dystrophy. We analysed Dystrophin binding dynamics in vivo for the first time. Within maturing fibres of host zebrafish embryos, our analysis reveals a pool of diffusible Dystrophin and complexes bound at the fibre membrane. Combining modelling, an improved FRAP methodology and direct semi-quantitative analysis of bleaching suggests the existence of two membrane-bound Dystrophin populations with widely differing bound lifetimes: a stable, tightly bound pool, and a dynamic bound pool with high turnover rate that exchanges with the cytoplasmic pool. The three populations were found consistently in human and zebrafish Dystrophins overexpressed in wild-type or dmdta222a/ta222a zebrafish embryos, which lack Dystrophin, and in Gt(dmd-Citrine)ct90a that express endogenously-driven tagged zebrafish Dystrophin. These results lead to a new model for Dystrophin membrane association in developing muscle, and highlight our methodology as a valuable strategy for in vivo analysis of complex protein dynamics. DOI: http://dx.doi.org/10.7554/eLife.06541.001 PMID:26459831

  5. Splice assembly tool and method of splicing

    DOEpatents

    Silva, Frank A.

    1980-01-01

    A splice assembly tool for assembling component parts of an electrical conductor while producing a splice connection between electrical cables therewith, comprises a first structural member adaptable for supporting force applying means thereon, said force applying means enabling a rotary force applied manually thereto to be converted to a longitudinal force for subsequent application against a first component part of said electrical connection, a second structural member adaptable for engaging a second component part in a manner to assist said first structural member in assembling the component parts relative to one another and transmission means for conveying said longitudinal force between said first and said second structural members, said first and said second structural members being coupled to one another by said transmission means, wherein at least one of said component parts comprises a tubular elastomeric sleeve and said force applying means provides a relatively high mechanical advantage when said rotary force is applied thereto so as to facilitate assembly of said at least one tubular elastomeric sleeve about said other component part in an interference fit manner.

  6. Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations.

    PubMed

    Maggio, Ignazio; Stefanucci, Luca; Janssen, Josephine M; Liu, Jin; Chen, Xiaoyu; Mouly, Vincent; Gonçalves, Manuel A F V

    2016-02-18

    Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles. PMID:26762977

  7. Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations

    PubMed Central

    Maggio, Ignazio; Stefanucci, Luca; Janssen, Josephine M.; Liu, Jin; Chen, Xiaoyu; Mouly, Vincent; Gonçalves, Manuel A.F.V.

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles. PMID:26762977

  8. Role of Dystrophin in Airway Smooth Muscle Phenotype, Contraction and Lung Function

    PubMed Central

    Sharma, Pawan; Basu, Sujata; Mitchell, Richard W.; Stelmack, Gerald L.; Anderson, Judy E.; Halayko, Andrew J.

    2014-01-01

    Dystrophin links the transmembrane dystrophin-glycoprotein complex to the actin cytoskeleton. We have shown that dystrophin-glycoprotein complex subunits are markers for airway smooth muscle phenotype maturation and together with caveolin-1, play an important role in calcium homeostasis. We tested if dystrophin affects phenotype maturation, tracheal contraction and lung physiology. We used dystrophin deficient Golden Retriever dogs (GRMD) and mdx mice vs healthy control animals in our approach. We found significant reduction of contractile protein markers: smooth muscle myosin heavy chain (smMHC) and calponin and reduced Ca2+ response to contractile agonist in dystrophin deficient cells. Immunocytochemistry revealed reduced stress fibers and number of smMHC positive cells in dystrophin-deficient cells, when compared to control. Immunoblot analysis of Akt1, GSK3β and mTOR phosphorylation further revealed that downstream PI3K signaling, which is essential for phenotype maturation, was suppressed in dystrophin deficient cell cultures. Tracheal rings from mdx mice showed significant reduction in the isometric contraction to methacholine (MCh) when compared to genetic control BL10ScSnJ mice (wild-type). In vivo lung function studies using a small animal ventilator revealed a significant reduction in peak airway resistance induced by maximum concentrations of inhaled MCh in mdx mice, while there was no change in other lung function parameters. These data show that the lack of dystrophin is associated with a concomitant suppression of ASM cell phenotype maturation in vitro, ASM contraction ex vivo and lung function in vivo, indicating that a linkage between the DGC and the actin cytoskeleton via dystrophin is a determinant of the phenotype and functional properties of ASM. PMID:25054970

  9. Structural Basis of Neuronal Nitric-oxide Synthase Interaction with Dystrophin Repeats 16 and 17.

    PubMed

    Molza, Anne-Elisabeth; Mangat, Khushdeep; Le Rumeur, Elisabeth; Hubert, Jean-François; Menhart, Nick; Delalande, Olivier

    2015-12-01

    Duchenne muscular dystrophy is a lethal genetic defect that is associated with the absence of dystrophin protein. Lack of dystrophin protein completely abolishes muscular nitric-oxide synthase (NOS) function as a regulator of blood flow during muscle contraction. In normal muscles, nNOS function is ensured by its localization at the sarcolemma through an interaction of its PDZ domain with dystrophin spectrin-like repeats R16 and R17. Early studies suggested that repeat R17 is the primary site of interaction but ignored the involved nNOS residues, and the R17 binding site has not been described at an atomic level. In this study, we characterized the specific amino acids involved in the binding site of nNOS-PDZ with dystrophin R16-17 using combined experimental biochemical and structural in silico approaches. First, 32 alanine-scanning mutagenesis variants of dystrophin R16-17 indicated the regions where mutagenesis modified the affinity of the dystrophin interaction with the nNOS-PDZ. Second, using small angle x-ray scattering-based models of dystrophin R16-17 and molecular docking methods, we generated atomic models of the dystrophin R16-17·nNOS-PDZ complex that correlated well with the alanine scanning identified regions of dystrophin. The structural regions constituting the dystrophin interaction surface involve the A/B loop and the N-terminal end of helix B of repeat R16 and the N-terminal end of helix A' and a small fraction of helix B' and a large part of the helix C' of repeat R17. The interaction surface of nNOS-PDZ involves its main β-sheet and its specific C-terminal β-finger. PMID:26378238

  10. Milder course in Duchenne patients with nonsense mutations and no muscle dystrophin.

    PubMed

    Zatz, M; Pavanello, R C M; Lazar, M; Yamamoto, G L; Lourenço, N C V; Cerqueira, A; Nogueira, L; Vainzof, M

    2014-11-01

    Duchenne muscular dystrophy (DMD), a severe and lethal condition, is caused by the absence of muscle dystrophin. Therapeutic trials aiming at the amelioration of muscle function have been targeting the production of muscle dystrophin in affected Duchenne patients. However, how much dystrophin is required to rescue the DMD phenotype remains an open question. We have previously identified two exceptional golden retriever muscular dystrophy (GRMD) dogs with a milder course despite the total absence of muscle dystrophin. Here we report two unusual patients carrying nonsense mutations in the DMD gene and dystrophin deficiency but with an unexpectedly mild phenotype. Three reported polymorphisms, respectively in genes LTBP4, SPP1 and ACTN3 were excluded as possible DMD genetic modifiers in our patients. Finding the mechanisms that protect some rare patients and dogs from the deleterious effect of absent muscle dystrophin is of utmost importance and may lead to new avenues for treatment. Importantly, these observations indicate that it is possible to have a functional large muscle even without dystrophin. PMID:25047667

  11. Expression of human dystrophin following the transplantation of genetically modified mdx myoblasts.

    PubMed

    Moisset, P A; Gagnon, Y; Karpati, G; Tremblay, J P

    1998-10-01

    Transplantation of genetically modified autologous myoblasts has been proposed as a possible solution to avoid long-term use of immunosuppressive drugs. To determine the conditions to be used in this kind of approach for possible treatment of dystrophin deficiency, mdx myoblasts were infected at different multiplicities of infection (MOI or 0.01-1000) with an adenoviral vector containing a CMV promoter/enhancer driven 6.3 kb human dystrophin cDNA (minigene) and tested in vitro for transgene expression. In these cultures, dystrophin mRNA was found to be proportionate with increasing MOI. Primary myoblast cultures derived from transgenic mdx mice expressing beta-Gal under a muscle-specific promoter and showing high expression of the human mini-dystrophin transgene introduced by the adenoviral vector were grafted into anterior tibialis muscles of SCID mice. Ten and 24 days after transplantation, numerous muscle fibers expressing both human dystrophin and beta-Gal were detected throughout the mouse muscles by immunohistochemistry using an antibody specific for human dystrophin. The presence of the human mini-dystrophin mRNA was also detected by RT-PCR. These results demonstrate that three essential conditions in autologous myoblast transplantation can be achieved: (1) in vivo survival of at least some of the transduced myoblasts; (2) efficient fusion of these cells with the host muscle fibers; and (3) the high expression of the dystrophin transgene in situ. Furthermore, this article provides a novel RT-PCR-based technique to quantify the human dystrophin minigene expression in vitro and in vivo. PMID:9930339

  12. BUILDING ROBUST TRANSCRIPTOMES WITH MASTER SPLICING FACTORS

    PubMed Central

    Jangi, Mohini; Sharp, Phillip A.

    2014-01-01

    Coherent splicing networks arise from many discrete splicing decisions regulated in unison. Here, we examine the properties of robust, context-specific splicing networks. We propose that a subset of key splicing regulators, or “master splicing factors,” respond to environmental cues to establish and maintain tissue transcriptomes during development. PMID:25417102

  13. Splicing Wires Permanently With Explosives

    NASA Technical Reports Server (NTRS)

    Bement, Laurence J.; Kushnick, Anne C.

    1990-01-01

    Explosive joining process developed to splice wires by enclosing and metallurgically bonding wires within copper sheets. Joints exhibit many desirable characteristics, 100-percent conductivity and strength, no heat-induced annealing, no susceptibility to corrosion in contacts between dissimilar metals, and stability at high temperature. Used to join wires to terminals, as well as to splice wires. Applicable to telecommunications industry, in which millions of small wires spliced annually.

  14. Alternative RNA splicing and cancer

    PubMed Central

    Liu, Sali; Cheng, Chonghui

    2015-01-01

    Alternative splicing of pre-messenger RNA (mRNA) is a fundamental mechanism by which a gene can give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. With the recognition that alternative splicing occurs in nearly all human genes, its relationship with cancer-associated pathways has emerged as a rapidly growing field. In this review, we summarize recent findings that have implicated the critical role of alternative splicing in cancer and discuss current understandings of the mechanisms underlying dysregulated alternative splicing in cancer cells. PMID:23765697

  15. ERISdb: a database of plant splice sites and splicing signals.

    PubMed

    Szcześniak, Michał Wojciech; Kabza, Michał; Pokrzywa, Rafał; Gudyś, Adam; Makałowska, Izabela

    2013-02-01

    Splicing is one of the major contributors to observed spatiotemporal diversification of transcripts and proteins in metazoans. There are numerous factors that affect the process, but splice sites themselves along with the adjacent splicing signals are critical here. Unfortunately, there is still little known about splicing in plants and, consequently, further research in some fields of plant molecular biology will encounter difficulties. Keeping this in mind, we performed a large-scale analysis of splice sites in eight plant species, using novel algorithms and tools developed by us. The analyses included identification of orthologous splice sites, polypyrimidine tracts and branch sites. Additionally we identified putative intronic and exonic cis-regulatory motifs, U12 introns as well as splice sites in 45 microRNA genes in five plant species. We also provide experimental evidence for plant splice sites in the form of expressed sequence tag and RNA-Seq data. All the data are stored in a novel database called ERISdb and are freely available at http://lemur.amu.edu.pl/share/ERISdb/. PMID:23299413

  16. Distal mdx muscle groups exhibiting up-regulation of utrophin and rescue of dystrophin-associated glycoproteins exemplify a protected phenotype in muscular dystrophy

    NASA Astrophysics Data System (ADS)

    Dowling, Paul; Culligan, Kevin; Ohlendieck, Kay

    2002-02-01

    Unique unaffected skeletal muscle fibres, unlike necrotic torso and limb muscles, may pave the way for a more detailed understanding of the molecular pathogenesis of inherited neuromuscular disorders and help to develop new treatment strategies for muscular dystrophies. The sparing of extraocular muscle in Duchenne muscular dystrophy is mostly attributed to the special protective properties of extremely fast-twitching small-diameter fibres, but here we show that distal muscles also represent a particular phenotype that is more resistant to necrosis. Immunoblot analysis of membranes isolated from the well established dystrophic animal model mdx shows that, in contrast to dystrophic limb muscles, the toe musculature exhibits an up-regulation of the autosomal dystrophin homologue utrophin and a concomitant rescue of dystrophin-associated glycoproteins. Thus distal mdx muscle groups provide a cellular system that naturally avoids myofibre degeneration which might be useful in the search for naturally occurring compensatory mechanisms in inherited skeletal muscle diseases.

  17. Monitoring Alternative Splicing Changes in Arabidopsis Circadian Clock Genes.

    PubMed

    Simpson, Craig G; Fuller, John; Calixto, Cristiane P G; McNicol, Jim; Booth, Clare; Brown, John W S; Staiger, Dorothee

    2016-01-01

    Posttranscriptional control makes an important contribution to circadian regulation of gene expression. In higher plants, alternative splicing is particularly prevalent upon abiotic and biotic stress and in the circadian system. Here we describe in detail a high-resolution reverse transcription-PCR based panel (HR RT-PCR) to monitor alternative splicing events. The use of the panel allows the quantification of changes in the proportion of splice isoforms between different samples, e.g., different time points, different tissues, genotypes, ecotypes, or treatments. PMID:26867620

  18. Lack of Dystrophin Affects Bronchial Epithelium in mdx Mice.

    PubMed

    Morici, Giuseppe; Rappa, Francesca; Cappello, Francesco; Pace, Elisabetta; Pace, Andrea; Mudò, Giuseppa; Crescimanno, Grazia; Belluardo, Natale; Bonsignore, Maria R

    2016-10-01

    Mild exercise training may positively affect the course of Duchenne Muscular Dystrophy (DMD). Training causes mild bronchial epithelial injury in both humans and mice, but no study assessed the effects of exercise in mdx mice, a well known model of DMD. The airway epithelium was examined in mdx (C57BL/10ScSn-Dmdmdx) mice, and in wild type (WT, C57BL/10ScSc) mice either under sedentary conditions (mdx-SD, WT-SD) or during mild exercise training (mdx-EX, WT-EX). At baseline, and after 30 and 45 days of training (5 d/wk for 6 weeks), epithelial morphology and markers of regeneration, apoptosis, and cellular stress were assessed. The number of goblet cells in bronchial epithelium was much lower in mdx than in WT mice under all conditions. At 30 days, epithelial regeneration (PCNA positive cells) was higher in EX than SD animals in both groups; however, at 45 days, epithelial regeneration decreased in mdx mice irrespective of training, and the percentage of apoptotic (TUNEL positive) cells was higher in mdx-EX than in WT-EX mice. Epithelial expression of HSP60 (marker of stress) progressively decreased, and inversely correlated with epithelial apoptosis (r = -0.66, P = 0.01) only in mdx mice. Lack of dystrophin in mdx mice appears associated with defective epithelial differentiation, and transient epithelial regeneration during mild exercise training. Hence, lack of dystrophin might impair repair in bronchial epithelium, with potential clinical consequences in DMD patients. J. Cell. Physiol. 231: 2218-2223, 2016. © 2016 Wiley Periodicals, Inc. PMID:26868633

  19. RNA splicing regulated by RBFOX1 is essential for cardiac function in zebrafish.

    PubMed

    Frese, Karen S; Meder, Benjamin; Keller, Andreas; Just, Steffen; Haas, Jan; Vogel, Britta; Fischer, Simon; Backes, Christina; Matzas, Mark; Köhler, Doreen; Benes, Vladimir; Katus, Hugo A; Rottbauer, Wolfgang

    2015-08-15

    Alternative splicing is one of the major mechanisms through which the proteomic and functional diversity of eukaryotes is achieved. However, the complex nature of the splicing machinery, its associated splicing regulators and the functional implications of alternatively spliced transcripts are only poorly understood. Here, we investigated the functional role of the splicing regulator rbfox1 in vivo using the zebrafish as a model system. We found that loss of rbfox1 led to progressive cardiac contractile dysfunction and heart failure. By using deep-transcriptome sequencing and quantitative real-time PCR, we show that depletion of rbfox1 in zebrafish results in an altered isoform expression of several crucial target genes, such as actn3a and hug. This study underlines that tightly regulated splicing is necessary for unconstrained cardiac function and renders the splicing regulator rbfox1 an interesting target for investigation in human heart failure and cardiomyopathy. PMID:26116573

  20. RNA splicing regulated by RBFOX1 is essential for cardiac function in zebrafish

    PubMed Central

    Frese, Karen S.; Meder, Benjamin; Keller, Andreas; Just, Steffen; Haas, Jan; Vogel, Britta; Fischer, Simon; Backes, Christina; Matzas, Mark; Köhler, Doreen; Benes, Vladimir; Katus, Hugo A.; Rottbauer, Wolfgang

    2015-01-01

    ABSTRACT Alternative splicing is one of the major mechanisms through which the proteomic and functional diversity of eukaryotes is achieved. However, the complex nature of the splicing machinery, its associated splicing regulators and the functional implications of alternatively spliced transcripts are only poorly understood. Here, we investigated the functional role of the splicing regulator rbfox1 in vivo using the zebrafish as a model system. We found that loss of rbfox1 led to progressive cardiac contractile dysfunction and heart failure. By using deep-transcriptome sequencing and quantitative real-time PCR, we show that depletion of rbfox1 in zebrafish results in an altered isoform expression of several crucial target genes, such as actn3a and hug. This study underlines that tightly regulated splicing is necessary for unconstrained cardiac function and renders the splicing regulator rbfox1 an interesting target for investigation in human heart failure and cardiomyopathy. PMID:26116573

  1. Cognitive flexibility deficits in a mouse model for the absence of full-length dystrophin.

    PubMed

    Remmelink, E; Aartsma-Rus, A; Smit, A B; Verhage, M; Loos, M; van Putten, M

    2016-07-01

    Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder, caused by mutations in the DMD gene and the resulting lack of dystrophin. The DMD gene has seven promoters, giving rise to multiple full-length and shorter isoforms. Besides the expression of dystrophin in muscles, the majority of dystrophin isoforms is expressed in brain and dystrophinopathy can lead to cognitive deficits, including intellectual impairments and deficits in executive function. In contrast to the muscle pathology, the impact of the lack of dystrophin on the brain is not very well studied. Here, we study the behavioral consequences of a lack of full-length dystrophin isoforms in mdx mice, particularly with regard to domains of executive functions and anxiety. We observed a deficit in cognitive flexibility in mdx mice in the absence of motor dysfunction or general learning impairments using two independent behavioral tests. In addition, increased anxiety was observed, but its expression depended on the context. Overall, these results suggest that the absence of full-length dystrophin in mice has specific behavioral effects that compare well to deficits observed in DMD patients. PMID:27220066

  2. Alternative splicing and muscular dystrophy

    PubMed Central

    Pistoni, Mariaelena; Ghigna, Claudia; Gabellini, Davide

    2013-01-01

    Alternative splicing of pre-mRNAs is a major contributor to proteomic diversity and to the control of gene expression in higher eukaryotic cells. For this reasons, alternative splicing is tightly regulated in different tissues and developmental stages and its disruption can lead to a wide range of human disorders. The aim of this review is to focus on the relevance of alternative splicing for muscle function and muscle disease. We begin by giving a brief overview of alternative splicing, muscle-specific gene expression and muscular dystrophy. Next, to illustrate these concepts we focus on two muscular dystrophy, myotonic muscular dystrophy and facioscapulohumeral muscular dystrophy, both associated to disruption of splicing regulation in muscle. PMID:20603608

  3. Therapeutic targeting of splicing in cancer.

    PubMed

    Lee, Stanley Chun-Wei; Abdel-Wahab, Omar

    2016-09-01

    Recent studies have highlighted that splicing patterns are frequently altered in cancer and that mutations in genes encoding spliceosomal proteins, as well as mutations affecting the splicing of key cancer-associated genes, are enriched in cancer. In parallel, there is also accumulating evidence that several molecular subtypes of cancer are highly dependent on splicing function for cell survival. These findings have resulted in a growing interest in targeting splicing catalysis, splicing regulatory proteins, and/or specific key altered splicing events in the treatment of cancer. Here we present strategies that exist and that are in development to target altered dependency on the spliceosome, as well as aberrant splicing, in cancer. These include drugs to target global splicing in cancer subtypes that are preferentially dependent on wild-type splicing for survival, methods to alter post-translational modifications of splicing-regulating proteins, and strategies to modulate pathologic splicing events and protein-RNA interactions in cancer. PMID:27603132

  4. Hydrogen Peroxide Alters Splicing of Soluble Guanylyl Cyclase and Selectively Modulates Expression of Splicing Regulators in Human Cancer Cells

    PubMed Central

    Cote, Gilbert J.; Zhu, Wen; Thomas, Anthony; Martin, Emil; Murad, Ferid; Sharina, Iraida G.

    2012-01-01

    Background Soluble guanylyl cyclase (sGC) plays a central role in nitric oxide (NO)-mediated signal transduction in the cardiovascular, nervous and gastrointestinal systems. Alternative RNA splicing has emerged as a potential mechanism to modulate sGC expression and activity. C-α1 sGC is an alternative splice form that is resistant to oxidation-induced protein degradation and demonstrates preferential subcellular distribution to the oxidized environment of endoplasmic reticulum (ER). Methodology/Principal Findings Here we report that splicing of C-α1 sGC can be modulated by H2O2 treatment in BE2 neuroblastoma and MDA-MD-468 adenocarcinoma human cells. In addition, we show that the H2O2 treatment of MDA-MD-468 cells selectively decreases protein levels of PTBP1 and hnRNP A2/B1 splice factors identified as potential α1 gene splicing regulators by in silico analysis. We further demonstrate that down-regulation of PTBP1 by H2O2 occurs at the protein level with variable regulation observed in different breast cancer cells. Conclusions/Significance Our data demonstrate that H2O2 regulates RNA splicing to induce expression of the oxidation-resistant C-α1 sGC subunit. We also report that H2O2 treatment selectively alters the expression of key splicing regulators. This process might play an important role in regulation of cellular adaptation to conditions of oxidative stress. PMID:22911749

  5. Systemic Trans-splicing adeno-associated viral delivery efficiently transduces the heart of adult mdx mouse, a model for duchenne muscular dystrophy.

    PubMed

    Ghosh, Arkasubhra; Yue, Yongping; Shin, Jin-Hong; Duan, Dongsheng

    2009-11-01

    Trans-splicing adeno-associated viral (tsAAV) vectors hold great promise for delivering large therapeutic genes. One potential application is in the treatment of Duchenne muscular dystrophy (DMD). In this case, it is necessary to transduce whole body muscle. We demonstrated body-wide AAV-9 tsAAV transduction in normal neonatal mice. However, it was not clear whether such an approach would work in diseased mice. In this study we delivered the AAV-9 alkaline phosphatase (AP) tsAAV vector (3 x 10(12) vector genome particles per vector per mouse, tail vein injection) to 2-month-old mdx mice, the most widely used DMD model. Four months later, we observed widespread AP expression in the heart. It reached the same level as we have seen in normal neonatal puppy. Interestingly, myocardial transduction correlated with beta-myosin heavy chain expression but not with LamR, the putative AAV-9 receptor. AP expression was also detected in various skeletal muscles but at levels much lower than in normal newborn mice. Despite the existing inflammatory milieu, we did not see any appreciable increase in CD4(+) and CD8(+) T cells and macrophages in striated muscles after systemic tsAAV infection. In summary, our results have paved the way for tsAAV-mediated gene therapy for Duchenne cardiomyopathy. PMID:19627234

  6. Human α7 Integrin Gene (ITGA7) Delivered by Adeno-Associated Virus Extends Survival of Severely Affected Dystrophin/Utrophin-Deficient Mice.

    PubMed

    Heller, Kristin N; Montgomery, Chrystal L; Shontz, Kimberly M; Clark, K Reed; Mendell, Jerry R; Rodino-Klapac, Louise R

    2015-10-01

    Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene. It is the most common, severe childhood form of muscular dystrophy. We investigated an alternative to dystrophin replacement by overexpressing ITGA7 using adeno-associated virus (AAV) delivery. ITGA7 is a laminin receptor in skeletal muscle that, like the dystrophin-glycoprotein complex, links the extracellular matrix to the internal actin cytoskeleton. ITGA7 is expressed in DMD patients and overexpression does not elicit an immune response to the transgene. We delivered rAAVrh.74.MCK.ITGA7 systemically at 5-7 days of age to the mdx/utrn(-/-) mouse deficient for dystrophin and utrophin, a severe mouse model of DMD. At 8 weeks postinjection, widespread expression of ITGA7 was observed at the sarcolemma of multiple muscle groups following gene transfer. The increased expression of ITGA7 significantly extended longevity and reduced common features of the mdx/utrn(-/-) mouse, including kyphosis. Overexpression of α7 expression protected against loss of force following contraction-induced damage and increased specific force in the diaphragm and EDL muscles 8 weeks after gene transfer. Taken together, these results further support the use of α7 integrin as a potential therapy for DMD. PMID:26076707

  7. Splicing Express: a software suite for alternative splicing analysis using next-generation sequencing data

    PubMed Central

    Kroll, Jose E.; Kim, Jihoon; Ohno-Machado, Lucila

    2015-01-01

    Motivation. Alternative splicing events (ASEs) are prevalent in the transcriptome of eukaryotic species and are known to influence many biological phenomena. The identification and quantification of these events are crucial for a better understanding of biological processes. Next-generation DNA sequencing technologies have allowed deep characterization of transcriptomes and made it possible to address these issues. ASEs analysis, however, represents a challenging task especially when many different samples need to be compared. Some popular tools for the analysis of ASEs are known to report thousands of events without annotations and/or graphical representations. A new tool for the identification and visualization of ASEs is here described, which can be used by biologists without a solid bioinformatics background. Results. A software suite named Splicing Express was created to perform ASEs analysis from transcriptome sequencing data derived from next-generation DNA sequencing platforms. Its major goal is to serve the needs of biomedical researchers who do not have bioinformatics skills. Splicing Express performs automatic annotation of transcriptome data (GTF files) using gene coordinates available from the UCSC genome browser and allows the analysis of data from all available species. The identification of ASEs is done by a known algorithm previously implemented in another tool named Splooce. As a final result, Splicing Express creates a set of HTML files composed of graphics and tables designed to describe the expression profile of ASEs among all analyzed samples. By using RNA-Seq data from the Illumina Human Body Map and the Rat Body Map, we show that Splicing Express is able to perform all tasks in a straightforward way, identifying well-known specific events. Availability and Implementation.Splicing Express is written in Perl and is suitable to run only in UNIX-like systems. More details can be found at: http

  8. Efficient Restoration of the Dystrophin Gene Reading Frame and Protein Structure in DMD Myoblasts Using the CinDel Method.

    PubMed

    Iyombe-Engembe, Jean-Paul; Ouellet, Dominique L; Barbeau, Xavier; Rousseau, Joël; Chapdelaine, Pierre; Lagüe, Patrick; Tremblay, Jacques P

    2016-01-01

    The CRISPR/Cas9 system is a great revolution in biology. This technology allows the modification of genes in vitro and in vivo in a wide variety of living organisms. In most Duchenne muscular dystrophy (DMD) patients, expression of dystrophin (DYS) protein is disrupted because exon deletions result in a frame shift. We present here the CRISPR-induced deletion (CinDel), a new promising genome-editing technology to correct the DMD gene. This strategy is based on the use of two gRNAs targeting specifically exons that precede and follow the patient deletion in the DMD gene. This pair of gRNAs induced a precise large additional deletion leading to fusion of the targeted exons. Using an adequate pair of gRNAs, the deletion of parts of these exons and the intron separating them restored the DMD reading frame in 62% of the hybrid exons in vitro in DMD myoblasts and in vivo in electroporated hDMD/mdx mice. Moreover, adequate pairs of gRNAs also restored the normal spectrin-like repeat of the dystrophin rod domain; such restoration is not obtained by exon skipping or deletion of complete exons. The expression of an internally deleted DYS protein was detected following the formation of myotubes by the unselected, treated DMD myoblasts. Given that CinDel induces permanent reparation of the DMD gene, this treatment would not have to be repeated as it is the case for exon skipping induced by oligonucleotides. PMID:26812655

  9. Minimum Factorization Agreement of Spliced ESTs

    NASA Astrophysics Data System (ADS)

    Bonizzoni, Paola; Della Vedova, Gianluca; Dondi, Riccardo; Pirola, Yuri; Rizzi, Raffaella

    Producing spliced EST sequences is a fundamental task in the computational problem of reconstructing splice and transcript variants, a crucial step in the alternative splicing investigation. Now, given an EST sequence, there can be several spliced EST sequences associated to it, since the original EST sequences may have different alignments against wide genomic regions.

  10. Nanoplasmonic probes of RNA folding and assembly during pre-mRNA splicing

    NASA Astrophysics Data System (ADS)

    Nguyen, Anh H.; Lee, Jong Uk; Sim, Sang Jun

    2016-02-01

    RNA splicing plays important roles in transcriptome and proteome diversity. Herein, we describe the use of a nanoplasmonic system that unveils RNA folding and assembly during pre-mRNA splicing wherein the quantification of mRNA splice variants is not taken into account. With a couple of SERS-probes and plasmonic probes binding at the boundary sites of exon-2/intron-2 and intron-2/exon-3 of the pre-mature RNA of the β-globin gene, the splicing process brings the probes into the plasmonic bands. For plasmonic probes, a plasmon shift increase of ~29 nm, corresponding to intron removal and exon-2 and exon-3 connection to form the mRNA molecule, is measured by plasmonic coupling. The increased scattering intensity and surface-enhanced Raman scattering (SERS) fingerprinting reveal the clear dynamics of pre-mRNA splicing. Moreover, a time-resolved experiment of individual RNA molecules exhibited a successful splicing and an inhibited splicing event by 33 μM biflavonoid isoginkgetin, a general inhibitor of RNA splicing. The results suggest that the RNA splicing is successfully monitored with the nanoplasmonic system. Thus, this platform can be useful for studying RNA nanotechnology, biomolecular folding, alternative splicing, and maturation of microRNA.

  11. Designing oligo libraries taking alternative splicing into account

    NASA Astrophysics Data System (ADS)

    Shoshan, Avi; Grebinskiy, Vladimir; Magen, Avner; Scolnicov, Ariel; Fink, Eyal; Lehavi, David; Wasserman, Alon

    2001-06-01

    We have designed sequences for DNA microarrays and oligo libraries, taking alternative splicing into account. Alternative splicing is a common phenomenon, occurring in more than 25% of the human genes. In many cases, different splice variants have different functions, are expressed in different tissues or may indicate different stages of disease. When designing sequences for DNA microarrays or oligo libraries, it is very important to take into account the sequence information of all the mRNA transcripts. Therefore, when a gene has more than one transcript (as a result of alternative splicing, alternative promoter sites or alternative poly-adenylation sites), it is very important to take all of them into account in the design. We have used the LEADS transcriptome prediction system to cluster and assemble the human sequences in GenBank and design optimal oligonucleotides for all the human genes with a known mRNA sequence based on the LEADS predictions.

  12. Splicing: is there an alternative contribution to Parkinson's disease?

    PubMed

    La Cognata, Valentina; D'Agata, Velia; Cavalcanti, Francesca; Cavallaro, Sebastiano

    2015-10-01

    Alternative splicing is a crucial mechanism of gene expression regulation that enormously increases the coding potential of our genome and represents an intermediate step between messenger RNA (mRNA) transcription and protein posttranslational modifications. Alternative splicing occupies a central position in the development and functions of the nervous system. Therefore, its deregulation frequently leads to several neurological human disorders. In the present review, we provide an updated overview on the impact of alternative splicing in Parkinson's disease (PD), the second most common neurodegenerative disorder worldwide. We will describe the alternative splicing of major PD-linked genes by collecting the current evidences about this intricate and not carefully explored aspect. Assessing the role of this mechanism on PD pathobiology may represent a central step toward an improved understanding of this complex disease. PMID:25980689

  13. Determinants of Plant U12-Dependent Intron Splicing Efficiency

    PubMed Central

    Lewandowska, Dominika; Simpson, Craig G.; Clark, Gillian P.; Jennings, Nikki S.; Barciszewska-Pacak, Maria; Lin, Chiao-Feng; Makalowski, Wojciech; Brown, John W.S.; Jarmolowski, Artur

    2004-01-01

    Factors affecting splicing of plant U12-dependent introns have been examined by extensive mutational analyses in an in vivo tobacco (Nicotiana tabacum) protoplast system using introns from three different Arabidopsis thaliana genes: CBP20, GSH2, and LD. The results provide evidence that splicing efficiency of plant U12 introns depends on a combination of factors, including UA content, exon bridging interactions between the U12 intron and flanking U2-dependent introns, and exon splicing enhancer sequences (ESEs). Unexpectedly, all three plant U12 introns required an adenosine at the upstream purine position in the branchpoint consensus UCCUURAUY. The exon upstream of the LD U12 intron is a major determinant of its higher level of splicing efficiency and potentially contains two ESE regions. These results suggest that in plants, U12 introns represent a level at which expression of their host genes can be regulated. PMID:15100401

  14. Abnormalities of dystrophin, the sarcoglycans, and laminin alpha2 in the muscular dystrophies.

    PubMed Central

    Jones, K J; Kim, S S; North, K N

    1998-01-01

    Abnormalities of dystrophin, the sarcoglycans, and laminin alpha2 are responsible for a subset of the muscular dystrophies. In this study we aim to characterise the nature and frequency of abnormalities of these proteins in an Australian population and to formulate an investigative algorithm to aid in approaching the diagnosis of the muscular dystrophies. To reduce ascertainment bias, biopsies with dystrophic (n=131) and non-dystrophic myopathic (n=71) changes were studied with antibodies to dystrophin, alpha, beta, and gamma sarcoglycan, beta dystroglycan, and laminin alpha2, and results were correlated with clinical phenotype. Abnormalities of dystrophin, the sarcoglycans, or laminin alpha2 were present in 61/131 (47%) dystrophic biopsies and in 0/71 myopathic biopsies, suggesting that immunocytochemical study of dystrophin, the sarcoglycans, and laminin alpha2 may, in general, be restricted to patients with dystrophic biopsies. Two patients with mutations identified in gamma sarcoglycan had abnormal dystrophin (by immunocytochemistry and immunoblot), showing that abnormalities of dystrophin may be a secondary phenomenon. Therefore, biopsies should not be excluded from sarcoglycan analysis on the basis of abnormal dystrophin alone. The diagnostic yield was highest in those with severe, rapidly progressive limb-girdle weakness (92%). Laminin alpha2 deficiency was identified in 5/131 (4%) patients; 215 patients presented after infancy, indicating that abnormalities of laminin alpha2 are not limited to the congenital muscular dystrophy phenotype. Overall patterns of immunocytochemistry and immunoblotting provided a guide to mutation analysis and, on the basis of this study, we have formulated a diagnostic algorithm to guide the investigation of patients with muscular dystrophy. Images PMID:9610800

  15. Phosphorylation within the cysteine-rich region of dystrophin enhances its association with β-dystroglycan and identifies a potential novel therapeutic target for skeletal muscle wasting.

    PubMed

    Swiderski, Kristy; Shaffer, Scott A; Gallis, Byron; Odom, Guy L; Arnett, Andrea L; Scott Edgar, J; Baum, Dale M; Chee, Annabel; Naim, Timur; Gregorevic, Paul; Murphy, Kate T; Moody, James; Goodlett, David R; Lynch, Gordon S; Chamberlain, Jeffrey S

    2014-12-20

    Mutations in dystrophin lead to Duchenne muscular dystrophy, which is among the most common human genetic disorders. Dystrophin nucleates assembly of the dystrophin-glycoprotein complex (DGC), and a defective DGC disrupts an essential link between the intracellular cytoskeleton and the basal lamina, leading to progressive muscle wasting. In vitro studies have suggested that dystrophin phosphorylation may affect interactions with actin or syntrophin, yet whether this occurs in vivo or affects protein function remains unknown. Utilizing nanoflow liquid chromatography mass spectrometry, we identified 18 phosphorylated residues within endogenous dystrophin. Mutagenesis revealed that phosphorylation at S3059 enhances the dystrophin-dystroglycan interaction and 3D modeling utilizing the Rosetta software program provided a structural model for how phosphorylation enhances this interaction. These findings demonstrate that phosphorylation is a key mechanism regulating the interaction between dystrophin and the DGC and reveal that posttranslational modification of a single amino acid directly modulates the function of dystrophin. PMID:25082828

  16. Splicing Efficiently Couples Optical Fibers

    NASA Technical Reports Server (NTRS)

    Lutes, G. F.

    1985-01-01

    Method of splicing single-mode optical fibers results in very low transmission losses through joined fiber ends. Coupling losses between joined optical-fiber ends only 0.1 dB. Method needs no special operator training.

  17. Ex vivo gene editing of the dystrophin gene in muscle stem cells mediated by peptide nucleic acid single stranded oligodeoxynucleotides induces stable expression of dystrophin in a mouse model for Duchenne muscular dystrophy.

    PubMed

    Nik-Ahd, Farnoosh; Bertoni, Carmen

    2014-07-01

    Duchenne muscular dystrophy (DMD) is a fatal disease caused by mutations in the dystrophin gene, which result in the complete absence of dystrophin protein throughout the body. Gene correction strategies hold promise to treating DMD. Our laboratory has previously demonstrated the ability of peptide nucleic acid single-stranded oligodeoxynucleotides (PNA-ssODNs) to permanently correct single-point mutations at the genomic level. In this study, we show that PNA-ssODNs can target and correct muscle satellite cells (SCs), a population of stem cells capable of self-renewing and differentiating into muscle fibers. When transplanted into skeletal muscles, SCs transfected with correcting PNA-ssODNs were able to engraft and to restore dystrophin expression. The number of dystrophin-positive fibers was shown to significantly increase over time. Expression was confirmed to be the result of the activation of a subpopulation of SCs that had undergone repair as demonstrated by immunofluorescence analyses of engrafted muscles using antibodies specific to full-length dystrophin transcripts and by genomic DNA analysis of dystrophin-positive fibers. Furthermore, the increase in dystrophin expression detected over time resulted in a significant improvement in muscle morphology. The ability of transplanted cells to return into quiescence and to activate upon demand was confirmed in all engrafted muscles following injury. These results demonstrate the feasibility of using gene editing strategies to target and correct SCs and further establish the therapeutic potential of this approach to permanently restore dystrophin expression into muscle of DMD patients. PMID:24753122

  18. Monoclonal antibodies against the muscle-specific N-terminus of dystrophin: Characterization of dystrophin in a muscular dystrophy patient with a frameshift deletion of Exons 3-7

    SciTech Connect

    Thanh, L. T.; Man, N. thi; Morris, G.E. ); Love, D.R.; Davies, K.E. ); Helliwell, T.R. )

    1993-07-01

    The first three exons of the human muscle dystrophin gene were expressed as a [beta]-galactosidase fusion protein. 1-his protein was then used to prepare two monoclonal antibodies (mAbs) which react with native dystrophin on frozen muscle sections and with denatured dystrophin on western blots but which do not cross-react with the distrophin-related protein, utrophin. Both mAbs recognized dystrophin in muscular dystrophy (MD) patients with deletions of exon 3, and further mapping with 11 overlapping synthetic peptides showed that they both recognize an epitope encoded by the muscle-specific exon 1. Neither mAb recognizes the brain dystrophin isoform, confirming the prediction from mRNA data that this has a different N-terminus. One Becker MD patient with a frameshift deletion of exons 3-7 is shown to produce dystrophin which reacts with the N-terminal mAbs, as well as with mAbs which bind on the C-terminal side of the deletion. The data suggest that transcription begins at the normal muscle dystrophin promoter and that the normal reading frame is restored after the deletion. A number of mechanisms have been proposed for restoration of the reading frame after deletion of exons 3-7, but those which predict dystrophin with an abnormal N-terminus do not appear to be major mechanisms in this patient. 27 refs., 6 figs.

  19. Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division

    PubMed Central

    Dumont, Nicolas A.; Wang, Yu Xin; von Maltzahn, Julia; Pasut, Alessandra; Bentzinger, C. Florian; Brun, Caroline E.; Rudnicki, Michael A.

    2016-01-01

    Dystrophin is expressed in differentiated myofibers where it is required for sarcolemmal integrity, and loss-of-function mutations in its gene result in Duchenne Muscular Dystrophy (DMD), a disease characterized by progressive and severe skeletal muscle degeneration. Here we found that dystrophin is also highly expressed in activated muscle stem cells (also known as satellite cells) where it associates with the Ser/Thr kinase Mark2 (also known as Par1b), an important regulator of cell polarity. In the absence of dystrophin, expression of Mark2 protein is downregulated, resulting in the inability to polarize Pard3 to the opposite side of the cell. Consequently, the number of asymmetric divisions is strikingly reduced in dystrophin-deficient satellite cells, while also displaying a loss of polarity, abnormal division patterns including centrosome amplification, impaired mitotic spindle orientation, and prolonged cell divisions. Altogether, these intrinsic defects strongly reduce the generation of myogenic progenitors needed for proper muscle regeneration. Therefore, we conclude that dystrophin has an essential role in the regulation of satellite cell polarity and asymmetric division. Our findings indicate that muscle wasting in DMD is not only caused by myofiber fragility, but is also exacerbated by impaired regeneration due to intrinsic satellite cell dysfunction. PMID:26569381

  20. The biochemical and mass spectrometric profiling of the dystrophin complexome from skeletal muscle

    PubMed Central

    Murphy, Sandra; Ohlendieck, Kay

    2015-01-01

    The development of advanced mass spectrometric methodology has decisively enhanced the analytical capabilities for studies into the composition and dynamics of multi-subunit protein complexes and their associated components. Large-scale complexome profiling is an approach that combines the systematic isolation and enrichment of protein assemblies with sophisticated mass spectrometry-based identification methods. In skeletal muscles, the membrane cytoskeletal protein dystrophin of 427 kDa forms tight interactions with a variety of sarcolemmal, cytosolic and extracellular proteins, which in turn associate with key components of the extracellular matrix and the intracellular cytoskeleton. A major function of this enormous assembly of proteins, including dystroglycans, sarcoglycans, syntrophins, dystrobrevins, sarcospan, laminin and cortical actin, is postulated to stabilize muscle fibres during the physical tensions of continuous excitation-contraction-relaxation cycles. This article reviews the evidence from recent proteomic studies that have focused on the characterization of the dystrophin-glycoprotein complex and its central role in the establishment of the cytoskeleton-sarcolemma-matrisome axis. Proteomic findings suggest a close linkage of the core dystrophin complex with a variety of protein species, including tubulin, vimentin, desmin, annexin, proteoglycans and collagens. Since the almost complete absence of dystrophin is the underlying cause for X-linked muscular dystrophy, a more detailed understanding of the composition, structure and plasticity of the dystrophin complexome may have considerable biomedical implications. PMID:26793286

  1. Genomic organization of the mouse dystrobrevin gene: Comparative analysis with the dystrophin gene

    SciTech Connect

    Ambrose, H.J.; Blake, D.J.; Nawrotzki, R.A.; Davies, K.E.

    1997-02-01

    Dystrobrevin, the mammalian orthologue of the Torpedo 87-kDa postsynaptic protein, is a member of the dystrophin gene family with homology to the cysteine-rich carboxy-terminal domain of dystrophin. Torpedo dystrobrevin copurifies with the acetylcholine receptors and is thought to form a complex with dystrophin and syntrophin. This complex is also found at the sarcolemma in vertebrates and defines the cytoplasmic component of the dystrophin-associated protein complex. Previously we have cloned several dystrobrevin isoforms from mouse brain and muscle. Here we show that these transcripts are the products of a single gene located on proximal mouse chromosome 18. To investigate the diversity of dystrobrevin transcripts we have determined that the mouse dystrobrevin gene is organized into 24 coding exons that span between 130 and 170 kb at the genomic level. The gene encodes at least three distinct protein isoforms that are expressed in a tissue-specific manner. Interestingly, although there is only 27% amino acid identity between the homologous regions of dystrobrevin and dystrophin, the positions of 8 of the 15 exon-intron junctions are identical. 47 refs., 4 figs., 2 tabs.

  2. Parental source effect of inherited mutations in the dystrophin gene of mice and men

    SciTech Connect

    Kress, W.; Grimm, T.; Mueller, C.R.; Bittner, R.

    1994-09-01

    Skewed X-inactivation has been suspected the genetic cause for some manifesting female carriers of BMD and DMD. To test whether a parental source effect on the protein expression of the dystrophin gene exists, we have set up backcrosses of mdx mice to wild type strains, enabling us to study the effect of the well-defined origin of the mutation on the dystrophin expression. In skeletal muscle sections the immunohistological staining patterns of dystrophin antibodies were showing a significant difference in the proportion of dystrophin positive versus negative fibers, suggesting a lower expression of paternally inherited mdx mutations. These data are in concordance with the pyruvate kinase (PK) levels in the serum: PK levels were much higher when the mutation was of maternal origin as compared to PK levels in paternally derived mutations. In order to test this {open_quotes}paternal source effect{close_quotes} in humans, we checked obligatory carriers of Becker muscular dystrophy (BMD) for the origin of their mutations. Creatin kinase (CK) levels in 21 carriers with maternally derived mutations were compared to CK values from 8 heterozygotes with mutations of paternal origin: CK (mat) = 140.3 IU/1 versus CK (pat) = 48.6 IU/I. The difference is statistically significant at the 5% level. These observations suggest either a differential X-inactivation or an imprinting of the dystrophin gene in mice and men.

  3. Characterization of the Regulation of CD46 RNA Alternative Splicing.

    PubMed

    Tang, Sze Jing; Luo, Shufang; Ho, Jia Xin Jessie; Ly, Phuong Thao; Goh, Eling; Roca, Xavier

    2016-07-01

    Here we present a detailed analysis of the alternative splicing regulation of human CD46, which generates different isoforms with distinct functions. CD46 is a ubiquitous membrane protein that protects host cells from complement and plays other roles in immunity, autophagy, and cell adhesion. CD46 deficiency causes an autoimmune disorder, and this protein is also involved in pathogen infection and cancer. Before this study, the mechanisms of CD46 alternative splicing remained unexplored even though dysregulation of this process has been associated with autoimmune diseases. We proved that the 5' splice sites of CD46 cassette exons 7 and 8 encoding extracellular domains are defined by noncanonical mechanisms of base pairing to U1 small nuclear RNA. Next we characterized the regulation of CD46 cassette exon 13, whose inclusion or skipping generates different cytoplasmic tails with distinct functions. Using splicing minigenes, we identified multiple exonic and intronic splicing enhancers and silencers that regulate exon 13 inclusion via trans-acting splicing factors like PTBP1 and TIAL1. Interestingly, a common splicing activator such as SRSF1 appears to repress CD46 exon 13 inclusion. We also report that expression of CD46 mRNA isoforms is further regulated by non-sense-mediated mRNA decay and transcription speed. Finally, we successfully manipulated CD46 exon 13 inclusion using antisense oligonucleotides, opening up opportunities for functional studies of the isoforms as well as for therapeutics for autoimmune diseases. This study provides insight into CD46 alternative splicing regulation with implications for its function in the immune system and for genetic disease. PMID:27226545

  4. Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing.

    PubMed

    Selvanathan, Saravana P; Graham, Garrett T; Erkizan, Hayriye V; Dirksen, Uta; Natarajan, Thanemozhi G; Dakic, Aleksandra; Yu, Songtao; Liu, Xuefeng; Paulsen, Michelle T; Ljungman, Mats E; Wu, Cathy H; Lawlor, Elizabeth R; Üren, Aykut; Toretsky, Jeffrey A

    2015-03-17

    The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron-exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4-279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4-279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code. PMID:25737553

  5. Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

    PubMed Central

    Selvanathan, Saravana P.; Erkizan, Hayriye V.; Dirksen, Uta; Natarajan, Thanemozhi G.; Dakic, Aleksandra; Yu, Songtao; Liu, Xuefeng; Paulsen, Michelle T.; Ljungman, Mats E.; Wu, Cathy H.; Lawlor, Elizabeth R.; Üren, Aykut; Toretsky, Jeffrey A.

    2015-01-01

    The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron–exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4–279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4–279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code. PMID:25737553

  6. Three exonic mutations in polycystic kidney disease-2 gene (PKD2) alter splicing of its pre-mRNA in a minigene system.

    PubMed

    Gonzalez-Paredes, Francisco J; Ramos-Trujillo, Elena; Claverie-Martin, Felix

    2016-03-01

    Exonic mutations are usually classified as missense, synonymous or nonsense mutations, however, they can affect pre-mRNA splicing either by disrupting splice sites, by creating new ones or by changing splicing regulatory sequences. In this study, we examined 21 mutations of the PKD2 gene, encoding polycystin-2, previously reported as missense or synonymous for their possible effects on pre-mRNA splicing using bioinformatics tools. All these mutations except one have been identified in patients with autosomal dominant polycystic kidney disease, a common genetic disorder characterized by the development and progressive enlargement of cysts in the kidneys leading to end-stage renal disease. We selected 12 missense mutations and 1 synonymous variant for the minigene assay, and found that three, c.1532A>T (p.D511V), c.1716G>A (p.K572K) and c.2657A>G (p.D886G) caused alterations in pre-mRNA splicing. Mutation c.1532A>T resulted in skipping of exon 6 and incorporation of a defective exon lacking the 3' end, while c.1716G>A led to skipping of exon 7. Mutation c.2657A>G resulted in incorporation of an incomplete exon 14, which is in agreement with previous results obtained with the patient's lymphoblast RNA. Our findings should be taken into account with regard to the pathogenicity of these PKD2 exonic mutations. These results together with previous reports highlight the importance to evaluate the effects of exonic single nucleotide substitutions in autosomal dominant polycystic kidney disease. PMID:26692149

  7. Alternative Splicing of TAF6: Downstream Transcriptome Impacts and Upstream RNA Splice Control Elements

    PubMed Central

    Kamtchueng, Catherine; Stébenne, Marie-Éve; Delannoy, Aurélie; Wilhelm, Emmanuelle; Léger, Hélène; Benecke, Arndt G.; Bell, Brendan

    2014-01-01

    The TAF6δ pathway of apoptosis can dictate life versus death decisions independently of the status of p53 tumor suppressor. TAF6δ is an inducible pro-apoptotic subunit of the general RNA polymerase II (Pol II) transcription factor TFIID. Alternative splice site choice of TAF6δ has been shown to be a pivotal event in triggering death via the TAF6δ pathway, yet nothing is currently known about the mechanisms that promote TAF6δ splicing. Furthermore the transcriptome impact of the gain of function of TAF6δ versus the loss of function of the major TAF6α splice form remains undefined. Here we employ comparative microarray analysis to show that TAF6δ drives a transcriptome profile distinct from that resulting from depletion of TAF6α. To define the cis-acting RNA elements responsible for TAF6δ alternative splicing we performed a mutational analysis of a TAF6 minigene system. The data point to several new RNA elements that can modulate TAF6δ and also reveal a role for RNA secondary structure in the selection of TAF6δ. PMID:25025302

  8. Our trails and trials in the subsarcolemmal cytoskeleton network and muscular dystrophy researches in the dystrophin era.

    PubMed

    Ozawa, Eijiro

    2010-01-01

    In 1987, about 150 years after the discovery of Duchenne muscular dystrophy (DMD), its responsible gene, the dystrophin gene, was cloned by Kunkel. This was a new substance. During these 20 odd years after the cloning, our understanding on dystrophin as a component of the subsarcolemmal cytoskeleton networks and on the pathomechanisms of and experimental therapeutics for DMD has been greatly enhanced. During this paradigm change, I was fortunately able to work as an active researcher on its frontiers for 12 years. After we discovered that dystrophin is located on the cell membrane in 1988, we studied the architecture of dystrophin and dystrophin-associated proteins (DAPs) complex in order to investigate the function of dystrophin and pathomechanism of DMD. During the conduct of these studies, we came to consider that the dystrophin-DAP complex serves to transmembranously connect the subsarcolemmal cytoskeleton networks and basal lamina to protect the lipid bilayer. It then became our working hypothesis that injury of the lipid bilayer upon muscle contraction is the cause of DMD. During this process, we predicted that subunits of the sarcoglycan (SG) complex are responsible for respective types of DMD-like muscular dystrophy with autosomal recessive inheritance. Our prediction was confirmed to be true by many researchers including ourselves. In this review, I will try to explain what we observed and how we considered concerning the architecture and function of the dystrophin-DAP complex, and the pathomechanisms of DMD and related muscular dystrophies. PMID:20948175

  9. Physiological Characterization of Muscle Strength With Variable Levels of Dystrophin Restoration in mdx Mice Following Local Antisense Therapy

    PubMed Central

    Sharp, Paul S; Bye-a-Jee, Hema; Wells, Dominic J

    2011-01-01

    Antisense-induced exon skipping can restore the open reading frame, and thus correct the dystrophin deficiency that causes Duchenne muscular dystrophy (DMD), a lethal muscle wasting condition. Successful proof-of-principle in preclinical models has led to human clinical trials. However, it is still not known what percentage of dystrophin-positive fibers and what level of expression is necessary for functional improvement. This study directly address these key questions in the mdx mouse model of DMD. To achieve a significant variation in dystrophin expression, we locally administered into tibialis anterior muscles various doses of a phosphorodiamidate morpholino oligomer (PMO) designed to skip the mutated exon 23 from the mRNA of murine dystrophin. We found a highly significant correlation between the number of dystrophin-positive fibers and resistance to contraction-induced injury, with a minimum of 20% of dystrophin-positive fibers required for meaningful improvement. Furthermore, our results also indicate that a relatively low level of dystrophin expression in muscle fibers may have significant clinical benefits. In contrast, improvements in muscle force were not correlated with either the number of positive fibers or total dystrophin levels, which highlight the need to conduct appropriate functional assessments in preclinical testing using the mdx mouse. PMID:20924363

  10. The missing puzzle piece: splicing mutations

    PubMed Central

    Lewandowska, Marzena A

    2013-01-01

    Proper gene splicing is highly dependent on the correct recognition of exons. Among the elements allowing this process are the “cis” (conserved sequences) and “trans” (snRNP, splicing factors) elements. Splicing mutations are related with a number of genetic disorders and usually induce exon skipping, form new exon/intron boundaries or activate new cryptic exons as a result of alterations at donor/acceptor sites. They constitute more than 9% of the currently published mutations, but this value is highly underestimated as many of the potential mutations are located in the “cis” elements and should be confirmed experimentally. The most commonly detected splicing mutations are located at donor (5’) and acceptor (3’) sites. Mutations at the branch point are rare (only over a dozen are known to date), and are mostly searched and detected when no alteration has been detected in the sequenced exons and UTRs. Polypyrimidine tract mutations are equally rare. High throughput technologies, as well as traditional Sanger sequencing, allow detection of many changes in intronic sequences and intron/exon boundaries. However, the assessment whether a mutation affects exon recognition and results in a genetic disorder has to be conducted using molecular biology methods: in vitro transcription of the sequence of interest cloned into a plasmid, with and without alterations, or mutation analysis via a hybrid minigene system. Even though microarrays and new generation sequencing methods pose difficulties in detecting novel branch point mutations, these tools seem appropriate to expand the mutation detection panel especially for diagnostic purposes. PMID:24294354

  11. SpliceVista, a Tool for Splice Variant Identification and Visualization in Shotgun Proteomics Data*

    PubMed Central

    Zhu, Yafeng; Hultin-Rosenberg, Lina; Forshed, Jenny; Branca, Rui M. M.; Orre, Lukas M.; Lehtiö, Janne

    2014-01-01

    Alternative splicing is a pervasive process in eukaryotic organisms. More than 90% of human genes have alternatively spliced products, and aberrant splicing has been shown to be associated with many diseases. Current methods employed in the detection of splice variants include prediction by clustering of expressed sequence tags, exon microarray, and mRNA sequencing, all methods focusing on RNA-level information. There is a lack of tools for analyzing splice variants at the protein level. Here, we present SpliceVista, a tool for splice variant identification and visualization based on mass spectrometry proteomics data. SpliceVista retrieves gene structure and translated sequences from alternative splicing databases and maps MS-identified peptides to splice variants. The visualization module plots the exon composition of each splice variant and aligns identified peptides with transcript positions. If quantitative mass spectrometry data are used, SpliceVista plots the quantitative patterns for each peptide and provides users with the option to cluster peptides based on their quantitative patterns. SpliceVista can identify splice-variant-specific peptides, providing the possibility for variant-specific analysis. The tool was tested on two experimental datasets (PXD000065 and PXD000134). In A431 cells treated with gefitinib, 2983 splice-variant-specific peptides corresponding to 939 splice variants were identified. Through comparison of splice-variant-centric, protein-centric, and gene-centric quantification, several genes (e.g. EIF4H) were found to have differentially regulated splice variants after gefitinib treatment. The same discrepancy between protein-centric and splice-centric quantification was detected in the other dataset, in which induced pluripotent stem cells were compared with parental fibroblast and human embryotic stem cells. In addition, SpliceVista can be used to visualize novel splice variants inferred from peptide-level evidence. In summary, Splice

  12. SpliceVista, a tool for splice variant identification and visualization in shotgun proteomics data.

    PubMed

    Zhu, Yafeng; Hultin-Rosenberg, Lina; Forshed, Jenny; Branca, Rui M M; Orre, Lukas M; Lehtiö, Janne

    2014-06-01

    Alternative splicing is a pervasive process in eukaryotic organisms. More than 90% of human genes have alternatively spliced products, and aberrant splicing has been shown to be associated with many diseases. Current methods employed in the detection of splice variants include prediction by clustering of expressed sequence tags, exon microarray, and mRNA sequencing, all methods focusing on RNA-level information. There is a lack of tools for analyzing splice variants at the protein level. Here, we present SpliceVista, a tool for splice variant identification and visualization based on mass spectrometry proteomics data. SpliceVista retrieves gene structure and translated sequences from alternative splicing databases and maps MS-identified peptides to splice variants. The visualization module plots the exon composition of each splice variant and aligns identified peptides with transcript positions. If quantitative mass spectrometry data are used, SpliceVista plots the quantitative patterns for each peptide and provides users with the option to cluster peptides based on their quantitative patterns. SpliceVista can identify splice-variant-specific peptides, providing the possibility for variant-specific analysis. The tool was tested on two experimental datasets (PXD000065 and PXD000134). In A431 cells treated with gefitinib, 2983 splice-variant-specific peptides corresponding to 939 splice variants were identified. Through comparison of splice-variant-centric, protein-centric, and gene-centric quantification, several genes (e.g. EIF4H) were found to have differentially regulated splice variants after gefitinib treatment. The same discrepancy between protein-centric and splice-centric quantification was detected in the other dataset, in which induced pluripotent stem cells were compared with parental fibroblast and human embryotic stem cells. In addition, SpliceVista can be used to visualize novel splice variants inferred from peptide-level evidence. In summary, Splice

  13. Adeno-Associated Virus (AAV) Mediated Dystrophin Gene Transfer Studies and Exon Skipping Strategies for Duchenne Muscular Dystrophy (DMD).

    PubMed

    Kawecka, Klaudia; Theodoulides, Michael; Hasoglu, Yalin; Jarmin, Susan; Kymalainen, Hanna; Le-Heron, Anita; Popplewell, Linda; Malerba, Alberto; Dickson, George; Athanasopoulos, Takis

    2015-01-01

    Duchenne muscular dystrophy (DMD), an X-linked inherited musclewasting disease primarily affecting young boys with prevalence of between1:3,500- 1:5,000, is a rare genetic disease caused by defects in the gene for dystrophin. Dystrophin protein is critical to the stability of myofibers in skeletal and cardiac muscle. There is currently no cure available to ameliorate DMD and/or its patho-physiology. A number of therapeutic strategies including molecular-based therapeutics that replace or correct the missing or nonfunctional dystrophin protein have been devised to correct the patho-physiological consequences induced by dystrophin absence. We will review the current in vivo experimentation status (including preclinical models and clinical trials) for two of these approaches, namely: 1) Adeno-associated virus (AAV) mediated (micro) dystrophin gene augmentation/ supplementation and 2) Antisense oligonucleotide (AON)-mediated exon skipping strategies. PMID:26159373

  14. In vivo and in vitro correction of the mdx dystrophin gene nonsense mutation by short-fragment homologous replacement.

    PubMed

    Kapsa, R; Quigley, A; Lynch, G S; Steeper, K; Kornberg, A J; Gregorevic, P; Austin, L; Byrne, E

    2001-04-10

    Targeted genetic correction of mutations in cells is a potential strategy for treating human conditions that involve nonsense, missense, and transcriptional splice junction mutations. One method of targeted gene repair, single-stranded short-fragment homologous replacement (ssSFHR), has been successful in repairing the common deltaF508 3-bp microdeletion at the cystic fibrosis transmembrane conductance regulator (CFTR) locus in 1% of airway epithelial cells in culture. This study investigates in vitro and in vivo application of a double-stranded method variant of SFHR gene repair to the mdx mouse model of Duchenne muscular dystrophy (DMD). A 603-bp wild-type PCR product was used to repair the exon 23 C-to-T mdx nonsense transition at the Xp21.1 dys locus in cultured myoblasts and in tibialis anterior (TA) from male mdx mice. Multiple transfection and variation of lipofection reagent both improved in vitro SFHR efficiency, with successful conversion of mdx to wild-type nucleotide at the dys locus achieved in 15 to 20% of cultured loci and in 0.0005 to 0.1% of TA. The genetic correction of mdx myoblasts was shown to persist for up to 28 days in culture and for at least 3 weeks in TA. While a high frequency of in vitro gene repair was observed, the lipofection used here appeared to have adverse effects on subsequent cell viability and corrected cells did not express dystrophin transcript. With further improvements to in vitro and in vivo gene repair efficiencies, SFHR may find some application in DMD and other genetic neuromuscular disorders in humans. PMID:11426463

  15. The sarcoglycan-sarcospan complex localization in mouse retina is independent from dystrophins

    PubMed Central

    Fort, Patrice; Estrada, Francisco-Javier; Bordais, Agnès; Mornet, Dominique; Sahel, José-Alain; Picaud, Serge; Vargas, Haydeé Rosas; Coral-Vázquez, Ramón M.; Rendon, Alvaro

    2005-01-01

    The sarcoglycan–sarcospan (SG–SSPN) complex is part of the dystrophin-glycoprotein complex that has been extensively characterized in muscle. To establish the framework for functional studies of sarcoglycans in retina here, we quantified sarcoglycans mRNA levels with real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and performed immunohistochemistry to determine their cellular and subcellular distribution. We showed that the β-, δ-, γ-, ε-sarcoglycans and sarcospan are expressed in mouse retina. They are localized predominantly in the outer and the inner limiting membranes, probably in the Müller cells and also in the ganglion cells axons where the expression of dystrophins have never been reported. We also investigated the status of the sarcoglycans in the retina of mdx3cv mutant mice for all Duchene Muscular Dystrophy (DMD) gene products. The absence of dystrophin did not produce any change in the sarcoglycan–sarcospan components expression and distribution. PMID:15993965

  16. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy.

    PubMed

    Long, Chengzu; Amoasii, Leonela; Mireault, Alex A; McAnally, John R; Li, Hui; Sanchez-Ortiz, Efrain; Bhattacharyya, Samadrita; Shelton, John M; Bassel-Duby, Rhonda; Olson, Eric N

    2016-01-22

    CRISPR/Cas9-mediated genome editing holds clinical potential for treating genetic diseases, such as Duchenne muscular dystrophy (DMD), which is caused by mutations in the dystrophin gene. To correct DMD by skipping mutant dystrophin exons in postnatal muscle tissue in vivo, we used adeno-associated virus-9 (AAV9) to deliver gene-editing components to postnatal mdx mice, a model of DMD. Different modes of AAV9 delivery were systematically tested, including intraperitoneal at postnatal day 1 (P1), intramuscular at P12, and retro-orbital at P18. Each of these methods restored dystrophin protein expression in cardiac and skeletal muscle to varying degrees, and expression increased from 3 to 12 weeks after injection. Postnatal gene editing also enhanced skeletal muscle function, as measured by grip strength tests 4 weeks after injection. This method provides a potential means of correcting mutations responsible for DMD and other monogenic disorders after birth. PMID:26721683

  17. Heteroduplex analysis of the dystrophin gene: Application to point mutation and carrier detection

    SciTech Connect

    Prior, T.W.; Papp, A.C.; Snyder, P.J.; Sedra, M.S.; Western, L.M.; Bartolo, C.; Mendell, J.R.; Moxley, R.T.

    1994-03-01

    Approximately one-third of Duchenne muscular dystrophy patients have undefined mutations in the dystrophin gene. For carrier and prenatal studies in families without detectable mutations, the indirect restriction fragment length polymorphism linkage approach is used. Using a multiplex amplification and heteroduplex analysis of dystrophin exons, the authors identified nonsense mutations in two DMD patients. Although the nonsense mutations are predicted to severely truncate the dystrophin protein, both patients presented with mild clinical courses of the disease. As a result of identifying the mutation in the affected boys, direct carrier studies by heteroduplex analysis were extended to other relatives. The authors conclude that the technique is not only ideal for mutation detection but is also useful for diagnostic testing. 29 refs., 4 figs.

  18. Spliced-leader trans-splicing in freshwater planarians.

    PubMed

    Zayas, Ricardo M; Bold, Tyler D; Newmark, Phillip A

    2005-10-01

    trans-Splicing, in which a spliced-leader (SL) RNA is appended to the most 5' exon of independently transcribed pre-mRNAs, has been described in a wide range of eukaryotes, from protozoans to chordates. Here we describe trans-splicing in the freshwater planarian Schmidtea mediterranea, a free-living member of the phylum Platyhelminthes. Analysis of an expressed sequence tag (EST) collection from this organism showed that over 300 transcripts shared one of two approximately 35-base sequences (Smed SL-1 and SL-2) at their 5' ends. Examination of genomic sequences encoding representatives of these transcripts revealed that these shared sequences were transcribed elsewhere in the genome. RNA blot analysis, 5' and 3' rapid amplification of cDNA ends, as well as genomic sequence data showed that 42-nt SL sequences were derived from small RNAs of approximately 110 nt. Similar sequences were also found at the 5' ends of ESTs from the planarian Dugesia japonica. trans-Splicing has already been described in numerous representatives of the phylum Platyhelminthes (trematodes, cestodes, and polyclads); its presence in two representatives of the triclads supports the hypothesis that this mode of RNA processing is ancestral within this group. The upcoming complete genome sequence of S. mediterranea, combined with this animal's experimental accessibility and susceptibility to RNAi, provide another model organism in which to study the function of the still-enigmatic trans-splicing. PMID:15972844

  19. Structural and Functional Alterations of Skeletal Muscle Microvasculature in Dystrophin-Deficient mdx Mice.

    PubMed

    Latroche, Claire; Matot, Béatrice; Martins-Bach, Aurea; Briand, David; Chazaud, Bénédicte; Wary, Claire; Carlier, Pierre G; Chrétien, Fabrice; Jouvion, Grégory

    2015-09-01

    Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease, caused by an absence of dystrophin, inevitably leading to death. Although muscle lesions are well characterized, blood vessel alterations that may have a major impact on muscle regeneration remain poorly understood. Our aim was to elucidate alterations of the vascular network organization, taking advantage of Flk1(GFP/+) crossed with mdx mice (model for human DMD where all blood vessels express green fluorescent protein) and functional repercussions using in vivo nuclear magnetic resonance, combining arterial spin-labeling imaging of perfusion, and (31)P-spectroscopy of phosphocreatine kinetics. For the first time, our study focused on old (12-month-old) mdx mice, displaying marked chronic muscle lesions, similar to the lesions observed in human DMD, in comparison to young-adult (3-month-old) mdx mice displaying only mild muscle lesions with no fibrosis. By using an original approach combining a specific animal model, state-of-the-art histology/morphometry techniques, and functional nuclear magnetic resonance, we demonstrated that the microvascular system is almost normal in young-adult in contrast to old mdx mice, displaying marked microvessel alterations, and the functional repercussions on muscle perfusion and bioenergetics after a hypoxic stress vary depending on stage of pathology. This original approach clarifies disease evolution and paves the way for setting up new diagnostic markers or therapeutic strategies. PMID:26193666

  20. Effective detection of corrected dystrophin loci in mdx mouse myogenic precursors.

    PubMed

    Todaro, Marian; Quigley, Anita; Kita, Magdalena; Chin, Judy; Lowes, Kym; Kornberg, Andrew J; Cook, Mark J; Kapsa, Robert

    2007-08-01

    Targeted corrective gene conversion (TCGC) holds much promise as a future therapy for many hereditary diseases in humans. Mutation correction frequencies varying between 0.0001% and 40% have been reported using chimeraplasty, oligoplasty, triplex-forming oligonucleotides, and small corrective PCR amplicons (CPA). However, PCR technologies used to detect correction events risk either falsely indicating or greatly exaggerating the presence of corrected loci. This is a problem that is considerably exacerbated by attempted improvement of the TCGC system using high corrective nucleic acid (CNA) to nuclear ratios. Small fragment homologous replacement (SFHR)-mediated correction of the exon 23 dystrophin (DMD) gene mutation in the mdx mouse model of DMD has been used in this study to evaluate the effect of increasing CPA amounts. In these experiments, we detected extremely high levels of apparently corrected loci and determined that at higher CNA to nuclear ratios the extent of locus correction was highly exaggerated by residual CNA species in the nucleic acids extracted from the treated cells. This study describes a generic locus-specific detection protocol designed to eradicate residual CNA species and avoid the artifactual or exaggerated detection of gene correction. PMID:17394239

  1. Delayed Cardiomyopathy in Dystrophin Deficient mdx Mice Relies on Intrinsic Glutathione Resource

    PubMed Central

    Khouzami, Lara; Bourin, Marie-Claude; Christov, Christo; Damy, Thibaud; Escoubet, Brigitte; Caramelle, Philippe; Perier, Magali; Wahbi, Karim; Meune, Christophe; Pavoine, Catherine; Pecker, Françoise

    2010-01-01

    Oxidative stress contributes to the pathogenesis of Duchenne muscular dystrophy (DMD). Although they have been a model for DMD, mdx mice exhibit slowly developing cardiomyopathy. We hypothesized that disease process was delayed owing to the development of an adaptive mechanism against oxidative stress, involving glutathione synthesis. At 15 to 20 weeks of age, mdx mice displayed a 33% increase in blood glutathione levels compared with age-matched C57BL/6 mice. In contrast, cardiac glutathione content was similar in mdx and C57BL/6 mice as a result of the balanced increased expression of glutamate cysteine ligase catalytic and regulatory subunits ensuring glutathione synthesis in the mdx mouse heart, as well as increased glutathione peroxidase-1 using glutathione. Oral administration from 10 weeks of age of the glutamate cysteine ligase inhibitor, l-buthionine(S,R)-sulfoximine (BSO, 5 mmol/L), led to a 33% and 50% drop in blood and cardiac glutathione, respectively, in 15- to 20-week-old mdx mice. Moreover, 20-week-old BSO-treated mdx mice displayed left ventricular hypertrophy associated with diastolic dysfunction, discontinuities in β-dystroglycan expression, micronecrosis and microangiopathic injuries. Examination of the glutathione status in four DMD patients showed that three displayed systemic glutathione deficiency as well. In conclusion, low glutathione resource hastens the onset of cardiomyopathy linked to a defect in dystrophin in mdx mice. This is relevant to the glutathione deficiency that DMD patients may suffer. PMID:20696779

  2. Accurate quantification of dystrophin mRNA and exon skipping levels in duchenne muscular dystrophy.

    PubMed

    Spitali, Pietro; Heemskerk, Hans; Vossen, Rolf H A M; Ferlini, Alessandra; den Dunnen, Johan T; 't Hoen, Peter A C; Aartsma-Rus, Annemieke

    2010-09-01

    Antisense oligonucleotide (AON)-mediated exon skipping aimed at restoring the reading frame is a promising therapeutic approach for Duchenne muscular dystrophy that is currently tested in clinical trials. Numerous AONs have been tested in (patient-derived) cultured muscle cells and the mdx mouse model. The main outcome to measure AON efficiency is usually the exon-skipping percentage, though different groups use different methods to assess these percentages. Here, we compare a series of techniques to quantify exon skipping levels in AON-treated mdx mouse muscle. We compared densitometry of RT-PCR products on ethidium bromide-stained agarose gels, primary and nested RT-PCR followed by bioanalyzer analysis and melting curve analysis. The digital array system (Fluidigm) allows absolute quantification of skipped vs non-skipped transcripts and was used as a reference. Digital array results show that 1 ng of mdx gastrocnemius muscle-derived mRNA contains approximately 1100 dystrophin transcripts and that 665 transcripts are sufficient to determine exon-skipping levels. Quantification using bioanalyzer or densitometric analysis of primary PCR products resulted in values close to those obtained with digital array. The use of the same technique allows comparison between different groups working on exon skipping in the mdx mouse model. PMID:20458276

  3. Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma.

    PubMed

    Chamberlain, Jeffrey S; Metzger, Joseph; Reyes, Morayma; Townsend, DeWayne; Faulkner, John A

    2007-07-01

    Duchenne muscular dystrophy (DMD) is the most common, lethal genetic disorder of children. A number of animal models of muscular dystrophy exist, but the most effective model for characterizing the structural and functional properties of dystrophin and therapeutic interventions has been the mdx mouse. Despite the approximately 20 years of investigations of the mdx mouse, the impact of the disease on the life span of mdx mice and the cause of death remain unresolved. Consequently, a life span study of the mdx mouse was designed that included cohorts of male and female mdx and wild-type C57BL/10 mice housed under specific pathogen-free conditions with deaths restricted to natural causes and with examination of the carcasses for pathology. Compared with wild-type mice, both mdx male and female mice had reduced life spans and displayed a progressively dystrophic muscle histopathology. Surprisingly, old mdx mice were prone to develop muscle tumors that resembled the human form of alveolar rhabdomyosarcoma, a cancer associated with poor prognosis. Rhabdomyosarcomas have not been observed previously in nontransgenic mice. The results substantiate the mdx mouse as an important model system for studies of the pathogenesis of and potential remedies for DMD. PMID:17360850

  4. Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy.

    PubMed Central

    Nguyen, T M; Morris, G E

    1993-01-01

    The majority of mutations in Xp21-linked muscular dystrophy (MD) can be identified by PCR or Southern blotting, as deletions or duplications of groups of exons in the dystrophin gene, but it is not always possible to predict how much altered dystrophin, if any, will be produced. Use of exon-specific monoclonal antibodies (mAbs) on muscle biopsies from MD patients can, in principle, provide information on both the amount of altered dystrophin produced and, when dystrophin is present, the nature of the genetic deletion or point mutation. For this purpose, mAbs which recognize regions of dystrophin encoded by known exons and whose binding is unaffected by the absence of adjacent exons are required. To map mAbs to specific exons, random "libraries" of expressed dystrophin fragments were created by cloning DNAseI digestion fragments of a 4.3-kb dystrophin cDNA into a pTEX expression vector. The libraries were then used to locate the epitopes recognized by 48 mAbs to fragments of 25-60 amino acids within the 1,434-amino-acid dystrophin fragment used to produce the antibodies. This is sufficiently detailed to allow further refinement by using synthetic peptides and, in many cases, to identify the exon in the DMD (Duchenne MD) gene which encodes the epitope. To illustrate their use in dystrophin analysis, a Duchenne patient with a frameshift deletion of exons 42 and 43 makes a truncated dystrophin encoded by exons 1-41, and we now show that this can be detected in the sarcolemma by mAbs up to and including those specific for exon 41 epitopes but not by mAbs specific for exon 43 or later epitopes. Images Figure 4 Figure 1 PMID:7684887

  5. Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy

    SciTech Connect

    Nguyen thi Man; Morris, G.E. )

    1993-06-01

    The majority of mutations in Xp21-linked muscular dystrophy (MD) can be identified by PCR or Southern blotting, as deletions or duplications of groups of exons in the dystrophin gene, but it is not always possible to predict how much altered dystrophin, if any, will be produced. Use of exon-specific monoclonal antibodies (mAbs) on muscle biopsies from MD patients can, in principle, provide information on both the amount of altered dystrophin produced and, when dystrophin is present, the nature of the genetic deletion or point mutation. For this purpose, mAbs which recognize regions of dystrophin encoded by known exons and whose binding is unaffected by the absence of adjacent exons are required. To map mAbs to specific exons, random [open quotes]libraries[close quotes] of expressed dystrophin fragments were created by cloning DNAseI digestion fragments of a 4.3-kb dystrophin cDNA into a pTEX expression vector. The libraries were then used to locate the epitopes recognized by 48 mAbs to fragments of 25--60 amino acids within the 1,434-amino-acid dystrophin fragment used to produce the antibodies. This is sufficiently detailed to allow further refinement by using synthetic peptides and, in many cases, to identify the exon in the DMD (Duchenne MD) gene which encodes the epitope. To illustrate their use in dystrophin analysis, a Duchenne patient with a frameshift deletion of exons 42 and 43 makes a truncated dystrophin encoded by exons 1--41, and the authors now show that this can be detected in the sarcolemma by mAbs up to and including those specific for exon 41 epitopes but not by mAbs specific for exon 43 or later epitopes. 38 refs., 2 figs., 4 tabs.

  6. Methods for Characterization of Alternative RNA Splicing

    PubMed Central

    Harvey, Samuel E.; Cheng, Chonghui

    2016-01-01

    Quantification of alternative splicing to detect the abundance of differentially spliced isoforms of a gene in total RNA can be accomplished via RT-PCR using both quantitative real-time and semi-quantitative PCR methods. These methods require careful PCR primer design to ensure specific detection of particular splice isoforms. We also describe analysis of alternative splicing using a splicing “minigene” in mammalian cell tissue culture to facilitate investigation of the regulation of alternative splicing of a particular exon of interest. PMID:26721495

  7. Detection of New Paternal Dystrophin Gene Mutations in Isolated Cases of Dystrophinopathy in Females

    PubMed Central

    Pegoraro, Elena; Schimke, R. Neil; Arahata, Kiichi; Hayashi, Yukiko; Stern, Harvey; Marks, Harold; Glasberg, Mark R.; Carroll, James E.; Taber, Joseph W.; Wessel, Henry B.; Bauserman, Steven C.; Marks, Warren A.; Toriello, Helga V.; Higgins, James V.; Appleton, Staci; Schwartz, Lisa; Garcia, Carlos A.; Hoffman, Eric P.

    1994-01-01

    Duchenne muscular dystrophy is one of the most common lethal monogenic disorders and is caused by dystrophin deficiency. The disease is transmitted as an X-linked recessive trait; however, recent biochemical and clinical studies have shown that many girls and women with a primary myopathy have an underlying dystrophinopathy, despite a negative family history for Duchenne dystrophy. These isolated female dystrophinopathy patients carried ambiguous diagnoses with presumed autosomal recessive inheritance (limbgirdle muscular dystrophy) prior to biochemical detection of dystrophin abnormalities in their muscle biopsy. It has been assumed that these female dystrophinopathy patients are heterozygous carriers who show preferential inactivation of the X chromosome harboring the normal dystrophin gene, although this has been shown for only a few X:autosome translocations and for two cases of discordant monozygotic twin female carriers. Here we study X-inactivation patterns of 13 female dystrophinopathy patients—10 isolated cases and 3 cases with a positive family history for Duchenne dystrophy in males. We show that all cases have skewed X-inactivation patterns in peripheral blood DNA. Of the nine isolated cases informative in our assay, eight showed inheritance of the dystrophin gene mutation from the paternal germ line. Only a single case showed maternal inheritance. The 10-fold higher incidence of paternal transmission of dystrophin gene mutations in these cases is at 30-fold variance with Bayesian predictions and gene mutation rates. Thus, our results suggest some mechanistic interaction between new dystrophin gene mutations, paternal inheritance, and skewed X inactivation. Our results provide both empirical risk data and a molecular diagnostic test method, which permit genetic counseling and prenatal diagnosis of this new category of patients. ImagesFigure 1 PMID:8198142

  8. Detection of new paternal dystrophin gene mutations in isolated cases of dystrophinopathy in females

    SciTech Connect

    Pegoraro, E.; Wessel, H.B.; Schwartz, L.; Hoffman, E.P. ); Schimke, R.N. ); Arahata, Kiichi; Hayashi, Yukiko ); Stern, H. ); Marks, H. ); Glasberg, M.R. )

    1994-06-01

    Duchenne muscular dystrophy is one of the most common lethal monogenic disorders and is caused by dystrophin deficiency. The disease is transmitted as an X-linked recessive trait; however, recent biochemical and clinical studies have shown that many girls and women with a primary myopathy have an underlying dystrophinopathy, despite a negative family history for Duchenne dystrophy. These isolated female dystrophinopathy patients carried ambiguous diagnoses with presumed autosomal recessive inheritance (limb-girdle muscular dystrophy) prior to biochemical detection of dystrophin abnormalities in their muscle biopsy. It has been assumed that these female dystrophinopathy patients are heterozygous carries who show preferential inactivation of the X chromosome harboring the normal dystrophin gene, although this has been shown for only a few X:autosome translocations and for two cases of discordant monozygotic twin female carriers. Here the authors study X-inactivation patterns of 13 female dystrophinopathy patients - 10 isolated cases and 3 cases with a positive family history for Duchenne dystrophy in males. They show that all cases have skewed X-inactivation patterns in peripheral blood DNA. Of the nine isolated cases informative in the assay, eight showed inheritance of the dystrophin gene mutation from the paternal germ line. Only a single case showed maternal inheritance. The 10-fold higher incidence of paternal transmission of dystrophin gene mutations in these cases is at 30-fold variance with Bayesian predictions and gene mutation rates. Thus, the results suggest some mechanistic interaction between new dystrophin gene mutations, paternal inheritance, and skewed X inactivation. The results provide both empirical risk data and a molecular diagnostic test method, which permit genetic counseling and prenatal diagnosis of this new category of patients. 58 refs., 7 figs., 2 tabs.

  9. A pair of RNA binding proteins controls networks of splicing events contributing to specialization of neural cell types

    PubMed Central

    Norris, Adam D.; Gao, Shangbang; Norris, Megan L.; Ray, Debashish; Ramani, Arun K.; Fraser, Andrew G.; Morris, Quaid; Hughes, Timothy R.; Zhen, Mei; Calarco, John A.

    2014-01-01

    SUMMARY Alternative splicing is important for the development and function of the nervous system, but little is known about the differences in alternative splicing between distinct types of neurons. Furthermore, the factors that control cell-type-specific splicing and the physiological roles of these alternative isoforms are unclear. By monitoring alternative splicing at single cell resolution in Caenorhabditis elegans, we demonstrate that splicing patterns in different neurons are often distinct and highly regulated. We identify two conserved RNA binding proteins, UNC-75/CELF and EXC-7/Hu/ELAV, which regulate overlapping networks of splicing events in GABAergic and cholinergic neurons. We use the UNC-75 exon network to discover regulators of synaptic transmission and to identify unique roles for isoforms of UNC-64/Syntaxin, a protein required for synaptic vesicle fusion. Our results indicate that combinatorial regulation of alternative splicing in distinct neurons provides a mechanism to specialize metazoan nervous systems. PMID:24910101

  10. MapSplice: Accurate mapping of RNA-seq reads for splice junction discovery

    PubMed Central

    Wang, Kai; Singh, Darshan; Zeng, Zheng; Coleman, Stephen J.; Huang, Yan; Savich, Gleb L.; He, Xiaping; Mieczkowski, Piotr; Grimm, Sara A.; Perou, Charles M.; MacLeod, James N.; Chiang, Derek Y.; Prins, Jan F.; Liu, Jinze

    2010-01-01

    The accurate mapping of reads that span splice junctions is a critical component of all analytic techniques that work with RNA-seq data. We introduce a second generation splice detection algorithm, MapSplice, whose focus is high sensitivity and specificity in the detection of splices as well as CPU and memory efficiency. MapSplice can be applied to both short (<75 bp) and long reads (≥75 bp). MapSplice is not dependent on splice site features or intron length, consequently it can detect novel canonical as well as non-canonical splices. MapSplice leverages the quality and diversity of read alignments of a given splice to increase accuracy. We demonstrate that MapSplice achieves higher sensitivity and specificity than TopHat and SpliceMap on a set of simulated RNA-seq data. Experimental studies also support the accuracy of the algorithm. Splice junctions derived from eight breast cancer RNA-seq datasets recapitulated the extensiveness of alternative splicing on a global level as well as the differences between molecular subtypes of breast cancer. These combined results indicate that MapSplice is a highly accurate algorithm for the alignment of RNA-seq reads to splice junctions. Software download URL: http://www.netlab.uky.edu/p/bioinfo/MapSplice. PMID:20802226

  11. MapSplice: accurate mapping of RNA-seq reads for splice junction discovery.

    PubMed

    Wang, Kai; Singh, Darshan; Zeng, Zheng; Coleman, Stephen J; Huang, Yan; Savich, Gleb L; He, Xiaping; Mieczkowski, Piotr; Grimm, Sara A; Perou, Charles M; MacLeod, James N; Chiang, Derek Y; Prins, Jan F; Liu, Jinze

    2010-10-01

    The accurate mapping of reads that span splice junctions is a critical component of all analytic techniques that work with RNA-seq data. We introduce a second generation splice detection algorithm, MapSplice, whose focus is high sensitivity and specificity in the detection of splices as well as CPU and memory efficiency. MapSplice can be applied to both short (<75 bp) and long reads (≥ 75 bp). MapSplice is not dependent on splice site features or intron length, consequently it can detect novel canonical as well as non-canonical splices. MapSplice leverages the quality and diversity of read alignments of a given splice to increase accuracy. We demonstrate that MapSplice achieves higher sensitivity and specificity than TopHat and SpliceMap on a set of simulated RNA-seq data. Experimental studies also support the accuracy of the algorithm. Splice junctions derived from eight breast cancer RNA-seq datasets recapitulated the extensiveness of alternative splicing on a global level as well as the differences between molecular subtypes of breast cancer. These combined results indicate that MapSplice is a highly accurate algorithm for the alignment of RNA-seq reads to splice junctions. Software download URL: http://www.netlab.uky.edu/p/bioinfo/MapSplice. PMID:20802226

  12. Long-time dynamics through parallel trajectory splicing

    SciTech Connect

    Perez, Danny; Cubuk, Ekin D.; Waterland, Amos; Kaxiras, Efthimios; Voter, Arthur F.

    2015-11-24

    Simulating the atomistic evolution of materials over long time scales is a longstanding challenge, especially for complex systems where the distribution of barrier heights is very heterogeneous. Such systems are difficult to investigate using conventional long-time scale techniques, and the fact that they tend to remain trapped in small regions of configuration space for extended periods of time strongly limits the physical insights gained from short simulations. We introduce a novel simulation technique, Parallel Trajectory Splicing (ParSplice), that aims at addressing this problem through the timewise parallelization of long trajectories. The computational efficiency of ParSplice stems from a speculation strategy whereby predictions of the future evolution of the system are leveraged to increase the amount of work that can be concurrently performed at any one time, hence improving the scalability of the method. ParSplice is also able to accurately account for, and potentially reuse, a substantial fraction of the computational work invested in the simulation. We validate the method on a simple Ag surface system and demonstrate substantial increases in efficiency compared to previous methods. As a result, we then demonstrate the power of ParSplice through the study of topology changes in Ag42Cu13 core–shell nanoparticles.

  13. Long-time dynamics through parallel trajectory splicing

    DOE PAGESBeta

    Perez, Danny; Cubuk, Ekin D.; Waterland, Amos; Kaxiras, Efthimios; Voter, Arthur F.

    2015-11-24

    Simulating the atomistic evolution of materials over long time scales is a longstanding challenge, especially for complex systems where the distribution of barrier heights is very heterogeneous. Such systems are difficult to investigate using conventional long-time scale techniques, and the fact that they tend to remain trapped in small regions of configuration space for extended periods of time strongly limits the physical insights gained from short simulations. We introduce a novel simulation technique, Parallel Trajectory Splicing (ParSplice), that aims at addressing this problem through the timewise parallelization of long trajectories. The computational efficiency of ParSplice stems from a speculation strategymore » whereby predictions of the future evolution of the system are leveraged to increase the amount of work that can be concurrently performed at any one time, hence improving the scalability of the method. ParSplice is also able to accurately account for, and potentially reuse, a substantial fraction of the computational work invested in the simulation. We validate the method on a simple Ag surface system and demonstrate substantial increases in efficiency compared to previous methods. As a result, we then demonstrate the power of ParSplice through the study of topology changes in Ag42Cu13 core–shell nanoparticles.« less

  14. Alternative Splice in Alternative Lice.

    PubMed

    Tovar-Corona, Jaime M; Castillo-Morales, Atahualpa; Chen, Lu; Olds, Brett P; Clark, John M; Reynolds, Stuart E; Pittendrigh, Barry R; Feil, Edward J; Urrutia, Araxi O

    2015-10-01

    Genomic and transcriptomics analyses have revealed human head and body lice to be almost genetically identical; although con-specific, they nevertheless occupy distinct ecological niches and have differing feeding patterns. Most importantly, while head lice are not known to be vector competent, body lice can transmit three serious bacterial diseases; epidemictyphus, trench fever, and relapsing fever. In order to gain insights into the molecular bases for these differences, we analyzed alternative splicing (AS) using next-generation sequencing data for one strain of head lice and one strain of body lice. We identified a total of 3,598 AS events which were head or body lice specific. Exon skipping AS events were overrepresented among both head and body lice, whereas intron retention events were underrepresented in both. However, both the enrichment of exon skipping and the underrepresentation of intron retention are significantly stronger in body lice compared with head lice. Genes containing body louse-specific AS events were found to be significantly enriched for functions associated with development of the nervous system, salivary gland, trachea, and ovarian follicle cells, as well as regulation of transcription. In contrast, no functional categories were overrepresented among genes with head louse-specific AS events. Together, our results constitute the first evidence for transcript pool differences in head and body lice, providing insights into molecular adaptations that enabled human lice to adapt to clothing, and representing a powerful illustration of the pivotal role AS can play in functional adaptation. PMID:26169943

  15. Alternative Splice in Alternative Lice

    PubMed Central

    Tovar-Corona, Jaime M.; Castillo-Morales, Atahualpa; Chen, Lu; Olds, Brett P.; Clark, John M.; Reynolds, Stuart E.; Pittendrigh, Barry R.; Feil, Edward J.; Urrutia, Araxi O.

    2015-01-01

    Genomic and transcriptomics analyses have revealed human head and body lice to be almost genetically identical; although con-specific, they nevertheless occupy distinct ecological niches and have differing feeding patterns. Most importantly, while head lice are not known to be vector competent, body lice can transmit three serious bacterial diseases; epidemictyphus, trench fever, and relapsing fever. In order to gain insights into the molecular bases for these differences, we analyzed alternative splicing (AS) using next-generation sequencing data for one strain of head lice and one strain of body lice. We identified a total of 3,598 AS events which were head or body lice specific. Exon skipping AS events were overrepresented among both head and body lice, whereas intron retention events were underrepresented in both. However, both the enrichment of exon skipping and the underrepresentation of intron retention are significantly stronger in body lice compared with head lice. Genes containing body louse-specific AS events were found to be significantly enriched for functions associated with development of the nervous system, salivary gland, trachea, and ovarian follicle cells, as well as regulation of transcription. In contrast, no functional categories were overrepresented among genes with head louse-specific AS events. Together, our results constitute the first evidence for transcript pool differences in head and body lice, providing insights into molecular adaptations that enabled human lice to adapt to clothing, and representing a powerful illustration of the pivotal role AS can play in functional adaptation. PMID:26169943

  16. The dystrophin gene and cognitive function in the general population

    PubMed Central

    Vojinovic, Dina; Adams, Hieab HH; van der Lee, Sven J; Ibrahim-Verbaas, Carla A; Brouwer, Rutger; van den Hout, Mirjam CGN; Oole, Edwin; van Rooij, Jeroen; Uitterlinden, Andre; Hofman, Albert; van IJcken, Wilfred FJ; Aartsma-Rus, Annemieke; van Ommen, GertJan B; Ikram, M Arfan; van Duijn, Cornelia M; Amin, Najaf

    2015-01-01

    The aim of our study is to investigate whether single-nucleotide dystrophin gene (DMD) variants associate with variability in cognitive functions in healthy populations. The study included 1240 participants from the Erasmus Rucphen family (ERF) study and 1464 individuals from the Rotterdam Study (RS). The participants whose exomes were sequenced and who were assessed for various cognitive traits were included in the analysis. To determine the association between DMD variants and cognitive ability, linear (mixed) modeling with adjustment for age, sex and education was used. Moreover, Sequence Kernel Association Test (SKAT) was used to test the overall association of the rare genetic variants present in the DMD with cognitive traits. Although no DMD variant surpassed the prespecified significance threshold (P<1 × 10−4), rs147546024:A>G showed strong association (β=1.786, P-value=2.56 × 10−4) with block-design test in the ERF study, while another variant rs1800273:G>A showed suggestive association (β=−0.465, P-value=0.002) with Mini-Mental State Examination test in the RS. Both variants are highly conserved, although rs147546024:A>G is an intronic variant, whereas rs1800273:G>A is a missense variant in the DMD which has a predicted damaging effect on the protein. Further gene-based analysis of DMD revealed suggestive association (P-values=0.087 and 0.074) with general cognitive ability in both cohorts. In conclusion, both single variant and gene-based analyses suggest the existence of variants in the DMD which may affect cognitive functioning in the general populations. PMID:25227141

  17. Alternatively Spliced Androgen Receptor Variants

    PubMed Central

    Dehm, Scott M.; Tindall, Donald J.

    2011-01-01

    Alternative splicing is an important mechanism for increasing functional diversity from a limited set of genes. De-regulation of this process is common in diverse pathologic conditions. The androgen receptor (AR) is a steroid receptor transcription factor with functions critical for normal male development as well as the growth and survival of normal and cancerous prostate tissue. Studies of AR function in androgen insensitivity syndrome (AIS) and prostate cancer (PCa) have demonstrated loss-of-function AR alterations in AIS, and gain-of-function AR alterations in PCa. Over the past two decades, AR gene alterations have been identified in various individuals with AIS, which disrupt normal AR splicing patterns and yield dysfunctional AR protein variants. More recently, altered AR splicing patterns have been identified as a mechanism of PCa progression and resistance to androgen-depletion therapy. Several studies have described the synthesis of alternatively spliced transcripts encoding truncated AR isoforms that lack the ligand-binding domain, which is the ultimate target of androgen depletion. Many of these truncated AR isoforms function as constitutively active, ligand-independent transcription factors that can support androgen-independent expression of AR target genes, as well as the androgen-independent growth of PCa cells. In this review, we will summarize the various alternatively spliced AR variants that have been discovered, with a focus on their role and origin in the pathologic conditions of AIS and PCa. PMID:21778211

  18. A conserved intronic U1 snRNP-binding sequence promotes trans-splicing in Drosophila

    PubMed Central

    Gao, Jun-Li; Fan, Yu-Jie; Wang, Xiu-Ye; Zhang, Yu; Pu, Jia; Li, Liang; Shao, Wei; Zhan, Shuai; Hao, Jianjiang

    2015-01-01

    Unlike typical cis-splicing, trans-splicing joins exons from two separate transcripts to produce chimeric mRNA and has been detected in most eukaryotes. Trans-splicing in trypanosomes and nematodes has been characterized as a spliced leader RNA-facilitated reaction; in contrast, its mechanism in higher eukaryotes remains unclear. Here we investigate mod(mdg4), a classic trans-spliced gene in Drosophila, and report that two critical RNA sequences in the middle of the last 5′ intron, TSA and TSB, promote trans-splicing of mod(mdg4). In TSA, a 13-nucleotide (nt) core motif is conserved across Drosophila species and is essential and sufficient for trans-splicing, which binds U1 small nuclear RNP (snRNP) through strong base-pairing with U1 snRNA. In TSB, a conserved secondary structure acts as an enhancer. Deletions of TSA and TSB using the CRISPR/Cas9 system result in developmental defects in flies. Although it is not clear how the 5′ intron finds the 3′ introns, compensatory changes in U1 snRNA rescue trans-splicing of TSA mutants, demonstrating that U1 recruitment is critical to promote trans-splicing in vivo. Furthermore, TSA core-like motifs are found in many other trans-spliced Drosophila genes, including lola. These findings represent a novel mechanism of trans-splicing, in which RNA motifs in the 5′ intron are sufficient to bring separate transcripts into close proximity to promote trans-splicing. PMID:25838544

  19. The RNA binding protein RBM38 (RNPC1) regulates splicing during late erythroid differentiation.

    PubMed

    Heinicke, Laurie A; Nabet, Behnam; Shen, Shihao; Jiang, Peng; van Zalen, Sebastiaan; Cieply, Benjamin; Russell, J Eric; Xing, Yi; Carstens, Russ P

    2013-01-01

    Alternative pre-mRNA splicing is a prevalent mechanism in mammals that promotes proteomic diversity, including expression of cell-type specific protein isoforms. We characterized a role for RBM38 (RNPC1) in regulation of alternative splicing during late erythroid differentiation. We used an Affymetrix human exon junction (HJAY) splicing microarray to identify a panel of RBM38-regulated alternatively spliced transcripts. Using microarray databases, we noted high RBM38 expression levels in CD71(+) erythroid cells and thus chose to examine RBM38 expression during erythroid differentiation of human hematopoietic stem cells, detecting enhanced RBM38 expression during late erythroid differentiation. In differentiated erythroid cells, we validated a subset of RBM38-regulated splicing events and determined that RBM38 regulates activation of Protein 4.1R (EPB41) exon 16 during late erythroid differentiation. Using Epb41 minigenes, Rbm38 was found to be a robust activator of exon 16 splicing. To further address the mechanism of RBM38-regulated alternative splicing, a novel mammalian protein expression system, followed by SELEX-Seq, was used to identify a GU-rich RBM38 binding motif. Lastly, using a tethering assay, we determined that RBM38 can directly activate splicing when recruited to a downstream intron. Together, our data support the role of RBM38 in regulating alternative splicing during erythroid differentiation. PMID:24250749

  20. The RNA Binding Protein RBM38 (RNPC1) Regulates Splicing during Late Erythroid Differentiation

    PubMed Central

    Heinicke, Laurie A.; Nabet, Behnam; Shen, Shihao; Jiang, Peng; van Zalen, Sebastiaan; Cieply, Benjamin; Russell, J. Eric; Xing, Yi; Carstens, Russ P.

    2013-01-01

    Alternative pre-mRNA splicing is a prevalent mechanism in mammals that promotes proteomic diversity, including expression of cell-type specific protein isoforms. We characterized a role for RBM38 (RNPC1) in regulation of alternative splicing during late erythroid differentiation. We used an Affymetrix human exon junction (HJAY) splicing microarray to identify a panel of RBM38-regulated alternatively spliced transcripts. Using microarray databases, we noted high RBM38 expression levels in CD71+ erythroid cells and thus chose to examine RBM38 expression during erythroid differentiation of human hematopoietic stem cells, detecting enhanced RBM38 expression during late erythroid differentiation. In differentiated erythroid cells, we validated a subset of RBM38-regulated splicing events and determined that RBM38 regulates activation of Protein 4.1R (EPB41) exon 16 during late erythroid differentiation. Using Epb41 minigenes, Rbm38 was found to be a robust activator of exon 16 splicing. To further address the mechanism of RBM38-regulated alternative splicing, a novel mammalian protein expression system, followed by SELEX-Seq, was used to identify a GU-rich RBM38 binding motif. Lastly, using a tethering assay, we determined that RBM38 can directly activate splicing when recruited to a downstream intron. Together, our data support the role of RBM38 in regulating alternative splicing during erythroid differentiation. PMID:24250749

  1. Achieving targeted and quantifiable alteration of mRNA splicing with Morpholino oligos

    SciTech Connect

    Morcos, Paul A. . E-mail: pmorcos@gene-tools.com

    2007-06-29

    This work represents the first guide for using steric-block antisense oligos as tools for effective and targeted modification of RNA splicing. Comparison of several steric-block oligo types shows the properties of Morpholinos provide significant advantages over other potential splice-blocking oligos. The procedures and complications of designing effective splice-blocking Morpholino oligos are described. The design process requires complete pre-mRNA sequence for defining suitable targets, which usually generate specific predictable messengers. To validate the targeting procedure, the level and nature of transcript alteration is characterized by RT-PCR analysis of splice modification in a {beta}-globin splice model system. An oligo-walking study reveals that while U1 and U2 small nuclear RiboNucleoProtein (snRNP) binding sites are the most effective targets for blocking splicing, inclusion of these sites is not required to achieve effective splice modifications. The most effective targeting strategy employs simultaneously blocking snRNP binding sites and splice-junctions. The work presented here continues to be the basis for most of the successful Morpholino oligos designed for the worldwide research community to block RNA splicing.

  2. Polypurine sequences within a downstream exon function as a splicing enhancer

    SciTech Connect

    Tanaka, Kenji; Watakabe, Akiya; Shimura, Yoshiro

    1994-02-01

    We have previously shown that a purine-rich sequence located within exon M2 of the mouse immunoglobulin {mu} gene functions as a splicing enhancer, as judged by its ability to stimulate splicing of a distant upstream intron. This sequence element has been designated ERS (exon recognition sequence). In this study, we investigated the stimulatory effects of various ERS-like sequences, using the in vitro splicing system with HeLa cell nuclear extracts. Here, we show that purine-rich sequences of several natural exons that have previously been shown to be required for splicing function as a splicing enhancer like the ERS of the immunoglobulin {mu} gene. Moreover, even synthetic polypurine sequences had stimulatory effects on the upstream splicing. Evaluation of the data obtained from the analyses of both natural and synthetic purine-rich sequences shows that (i) alternating purine sequences can stimulate splicing, while poly(A) or poly(G) sequences cannot, and (ii) the presence of U residues within the polypurine sequence greatly reduces the level of stimulation. Competition experiments strongly suggest that the stimulatory effects of various purine-rich sequences are mediated by the same trans-acting factor(s). We conclude from these results that the purine-rich sequences that we examined in this study also represent examples of ERS. Thus, ERS is considered a general splicing element that is present in various exons and plays an important role in splice site selection. 50 refs., 7 figs., 2 tabs.

  3. An intronic LINE-1 element insertion in the dystrophin gene aborts dystrophin expression and results in Duchenne-like muscular dystrophy in the corgi breed.

    PubMed

    Smith, Bruce F; Yue, Yongping; Woods, Philip R; Kornegay, Joe N; Shin, Jin-Hong; Williams, Regina R; Duan, Dongsheng

    2011-02-01

    Duchenne muscular dystrophy (DMD) is a dystrophin-deficient lethal muscle disease. To date, the catastrophic muscle wasting phenotype has only been seen in dystrophin-deficient humans and dogs. Although Duchenne-like symptoms have been observed in more than a dozen dog breeds, the mutation is often not known and research colonies are rarely established. Here, we report an independent canine DMD model originally derived from the Pembroke Welsh corgi breed. The affected dogs presented clinical signs of muscular dystrophy. Immunostaining revealed the absence of dystrophin and upregulation of utrophin. Histopathologic examination showed variable fiber size, central nucleation, calcification, fibrosis, neutrophil and macrophage infiltration and cardiac focal vacuolar degeneration. Carrier dogs also displayed mild myopathy. The mutation was identified as a long interspersed repetitive element-1 (LINE-1) insertion in intron 13, which introduced a new exon containing an in-frame stop codon. Similar mutations have been seen in human patients. A colony was generated by crossing carrier females with normal males. Affected puppies had a normal birth weight but they experienced a striking growth delay in the first 5 days. In summary, the new corgi DMD model offers an excellent opportunity to study DMD pathogenesis and to develop novel therapies. PMID:20714321

  4. Environmental qualification of heat shrinkable splices using the degradation factor method

    SciTech Connect

    Allman, M.J.; Smith, D.L.

    1989-03-01

    The environmental qualification of heat shrinkable splices is investigated with respect to abnormal installation and its effect on insulation resistance. An analytical method is developed to assess the installed splice insulation resistance during a design basis accident at a nuclear facility. Once the insulation resistance is established a system's performance capabilities can also be verified analytically.

  5. Diverse alternative back-splicing and alternative splicing landscape of circular RNAs.

    PubMed

    Zhang, Xiao-Ou; Dong, Rui; Zhang, Yang; Zhang, Jia-Lin; Luo, Zheng; Zhang, Jun; Chen, Ling-Ling; Yang, Li

    2016-09-01

    Circular RNAs (circRNAs) derived from back-spliced exons have been widely identified as being co-expressed with their linear counterparts. A single gene locus can produce multiple circRNAs through alternative back-splice site selection and/or alternative splice site selection; however, a detailed map of alternative back-splicing/splicing in circRNAs is lacking. Here, with the upgraded CIRCexplorer2 pipeline, we systematically annotated different types of alternative back-splicing and alternative splicing events in circRNAs from various cell lines. Compared with their linear cognate RNAs, circRNAs exhibited distinct patterns of alternative back-splicing and alternative splicing. Alternative back-splice site selection was correlated with the competition of putative RNA pairs across introns that bracket alternative back-splice sites. In addition, all four basic types of alternative splicing that have been identified in the (linear) mRNA process were found within circRNAs, and many exons were predominantly spliced in circRNAs. Unexpectedly, thousands of previously unannotated exons were detected in circRNAs from the examined cell lines. Although these novel exons had similar splice site strength, they were much less conserved than known exons in sequences. Finally, both alternative back-splicing and circRNA-predominant alternative splicing were highly diverse among the examined cell lines. All of the identified alternative back-splicing and alternative splicing in circRNAs are available in the CIRCpedia database (http://www.picb.ac.cn/rnomics/circpedia). Collectively, the annotation of alternative back-splicing and alternative splicing in circRNAs provides a valuable resource for depicting the complexity of circRNA biogenesis and for studying the potential functions of circRNAs in different cells. PMID:27365365

  6. The Role of Alternative Splicing in the Control of Immune Homeostasis and Cellular Differentiation.

    PubMed

    Yabas, Mehmet; Elliott, Hannah; Hoyne, Gerard F

    2016-01-01

    Alternative splicing of pre-mRNA helps to enhance the genetic diversity within mammalian cells by increasing the number of protein isoforms that can be generated from one gene product. This provides a great deal of flexibility to the host cell to alter protein function, but when dysregulation in splicing occurs this can have important impact on health and disease. Alternative splicing is widely used in the mammalian immune system to control the development and function of antigen specific lymphocytes. In this review we will examine the splicing of pre-mRNAs yielding key proteins in the immune system that regulate apoptosis, lymphocyte differentiation, activation and homeostasis, and discuss how defects in splicing can contribute to diseases. We will describe how disruption to trans-acting factors, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), can impact on cell survival and differentiation in the immune system. PMID:26703587

  7. The Role of Alternative Splicing in the Control of Immune Homeostasis and Cellular Differentiation

    PubMed Central

    Yabas, Mehmet; Elliott, Hannah; Hoyne, Gerard F.

    2015-01-01

    Alternative splicing of pre-mRNA helps to enhance the genetic diversity within mammalian cells by increasing the number of protein isoforms that can be generated from one gene product. This provides a great deal of flexibility to the host cell to alter protein function, but when dysregulation in splicing occurs this can have important impact on health and disease. Alternative splicing is widely used in the mammalian immune system to control the development and function of antigen specific lymphocytes. In this review we will examine the splicing of pre-mRNAs yielding key proteins in the immune system that regulate apoptosis, lymphocyte differentiation, activation and homeostasis, and discuss how defects in splicing can contribute to diseases. We will describe how disruption to trans-acting factors, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), can impact on cell survival and differentiation in the immune system. PMID:26703587

  8. Dystrophin and Dysferlin Double Mutant Mice: A Novel Model For Rhabdomyosarcoma

    PubMed Central

    Hosur, Vishnu; Kavirayani, Anoop; Riefler, Jennifer; Carney, Lisa M.B.; Lyons, Bonnie; Gott, Bruce; Cox, Gregory A.; Shultz, Leonard D.

    2012-01-01

    While researchers are yet to establish a link a between muscular dystrophy (MD) and sarcomas in human patients, literature suggests that MD genes dystrophin and dysferlin act as tumor suppressor genes in mouse models of MD. For instance, dystrophin deficient mdx and dysferlin deficient A/J mice, models of human Duchenne Muscular Dystrophy and Limb Girdle Muscular Dystrophy type 2B, respectively, develop mixed sarcomas with variable penetrance and latency. To further establish the correlation between MD and sarcoma development, and to test whether a combined deletion of dystrophin and dysferlin exacerbates MD and augments the incidence of sarcomas, we generated dystrophin and dysferlin double mutant mice (STOCK-Dysfprmd Dmdmdx-5Cv). Not surprisingly, the double mutant mice develop severe MD symptoms and moreover develop rhabdomyosarcoma at an average age of 12 months, with an incidence of > 90%. Histological and immunohistochemical analyses, using a panel of antibodies against skeletal muscle cell proteins, electron microscopy, cytogenetics, and molecular analysis reveal that the double mutant mice develop rhabdomyosarcoma. The present finding bolsters the correlation between MD and sarcomas, and provides a model not only to examine the cellular origins but also to identify mechanisms and signal transduction pathways triggering development of RMS. PMID:22682622

  9. Dystrophin and the two related genetic diseases, Duchenne and Becker muscular dystrophies

    PubMed Central

    Rumeur, Elisabeth Le

    2015-01-01

    Mutations of the dystrophin DMD gene, essentially deletions of one or several exons, are the cause of two devastating and to date incurable diseases, Duchenne (DMD) and Becker (BMD) muscular dystrophies. Depending upon the preservation or not of the reading frame, dystrophin is completely absent in DMD, or present in either a mutated or a truncated form in BMD. DMD is a severe disease which leads to a premature death of the patients. Therapy approaches are evolving with the aim to transform the severe DMD in the BMD form of the disease by restoring the expression of a mutated or truncated dystrophin. These therapies are based on the assumption that BMD is a mild disease. However, this is not completely true as BMD patients are more or less severely affected and no molecular basis of this heterogeneity of the BMD form of the disease is yet understood. The aim of this review is to report for the correlation between dystrophin structures in BMD deletions in view of this heterogeneity and to emphasize that examining BMD patients in details is highly relevant to anticipate for DMD therapy effects. PMID:26295289

  10. Study about locomotory ability of dystrophin-defected C.elegans after spaceflight

    NASA Astrophysics Data System (ADS)

    Gao, Ying; Sun, Yeqing; Lei, Huang; Xu, Dan

    2012-07-01

    Space microgravity could induce a variety of biological changes such as muscular atrophy. Recent studies show that gravisensing is a key point in muscular atrophy process, but the molecular mechanism is still unknown. Dystrophin, a muscle-related protein, plays an important role in muscle development. It is reported that mutation of human dystrophin gene could cause muscular atrophy. In this study, we focus on whether dystrophin gene acts as a gravisensing factor and observe locomotory ability of dystrophin-defected Caenorhabditis elegans (C.elegans) after spaceflight. We used wild-type (WT) and dystrophin-defected (dys-1) mutant of C.elegans, which were cultured to dauer stage and sent to space by Shenzhou 8 spacecraft (from Nov 1st to 17th, 2011). These worms were divided into three groups: space group (space radiation and microgravity conditions), space control group (space radiation and chmetcnvTCSC0NumberType1NegativeFalseHasSpaceFalseSourceValue1UnitNameg1g centrifuge force conditions) and ground control group.We already observed the progeny (generation F1 and F2) of worms which were sent to space, the movement of C. elegans is restricted to a two-dimensional sinusoidal pattern, and evaluated locomotory ability by the ratio (length/width) in crawl trace wave of C. elegans. The increased value of ratio indicates the decrease in locomotory ability of C. elegans. Our results from generation F1 showed that WT worms in space group(7.7±1.8) demonstrated the significant decrease in locomotory ability about 15%, compared with those in space control group(6.7±1.2). This finding indicates that locomotory ability of C. elegans progeny could be affected by microgravity in space environment. In comparison to the obvious difference in ratio between space group and space control group for WT worms, there is no significant difference between two space groups of generation F2 .For dys-1 mutant of C.elegans (generation F1 and F2), the results show that dystrophin deficiency

  11. Alternative splicing and expression profile analysis of expressed sequence tags in domestic pig.

    PubMed

    Zhang, Liang; Tao, Lin; Ye, Lin; He, Ling; Zhu, Yuan-Zhong; Zhu, Yue-Dong; Zhou, Yan

    2007-02-01

    Domestic pig (Sus scrofa domestica) is one of the most important mammals to humans. Alternative splicing is a cellular mechanism in eukaryotes that greatly increases the diversity of gene products. Expression sequence tags (ESTs) have been widely used for gene discovery, expression profile analysis, and alternative splicing detection. In this study, a total of 712,905 ESTs extracted from 101 different non-normalized EST libraries of the domestic pig were analyzed. These EST libraries cover the nervous system, digestive system, immune system, and meat production related tissues from embryo, newborn, and adult pigs, making contributions to the analysis of alternative splicing variants as well as expression profiles in various stages of tissues. A modified approach was designed to cluster and assemble large EST datasets, aiming to detect alternative splicing together with EST abundance of each splicing variant. Much efforts were made to classify alternative splicing into different types and apply different filters to each type to get more reliable results. Finally, a total of 1,223 genes with average 2.8 splicing variants were detected among 16,540 unique genes. The overview of expression profiles would change when we take alternative splicing into account. PMID:17572361

  12. Decreased stability and translation of T cell receptor zeta mRNA with an alternatively spliced 3'-untranslated region contribute to zeta chain down-regulation in patients with systemic lupus erythematosus.

    PubMed

    Chowdhury, Bhabadeb; Tsokos, Christos G; Krishnan, Sandeep; Robertson, James; Fisher, Carolyn U; Warke, Rahul G; Warke, Vishal G; Nambiar, Madhusoodana P; Tsokos, George C

    2005-05-13

    The molecular mechanisms involved in the aberrant expression of T cell receptor (TCR) zeta chain of patients with systemic lupus erythematosus are not known. Previously we demonstrated that although normal T cells express high levels of TCR zeta mRNA with wild-type (WT) 3' untranslated region (3' UTR), systemic lupus erythematosus T cells display significantly high levels of TCR zeta mRNA with the alternatively spliced (AS) 3' UTR form, which is derived by splice deletion of nucleotides 672-1233 of the TCR zeta transcript. Here we report that the stability of TCR zeta mRNA with an AS 3' UTR is low compared with TCR zeta mRNA with WT 3' UTR. AS 3' UTR, but not WT 3' UTR, conferred similar instability to the luciferase gene. Immunoblotting of cell lysates derived from transfected COS-7 cells demonstrated that TCR zeta with AS 3' UTR produced low amounts of 16-kDa protein. In vitro transcription and translation also produced low amounts of protein from TCR zeta with AS 3' UTR. Taken together our findings suggest that nucleotides 672-1233 bp of TCR zeta 3' UTR play a critical role in its stability and also have elements required for the translational regulation of TCR zeta chain expression in human T cells. PMID:15743765

  13. Early Progressive Dilated Cardiomyopathy in a Family with Becker Muscular Dystrophy Related to a Novel Frameshift Mutation in the Dystrophin Gene Exon 27

    PubMed Central

    Tsuda, Takeshi; Fitzgerald, Kristi; Scavena, Mena; Gidding, Samuel; Cox, Mary O.; Marks, Harold; Flanigan, Kevin M.; Moore, Steven A.

    2014-01-01

    We report a family in which two male siblings with Becker muscular dystrophy (BMD) developed severe dilated cardiomyopathy (DCM) and progressive heart failure (HF) at age 11; one died at age 14 years while awaiting heart transplant and the other underwent left ventricular assist device (LVAD) implantation at the same age. Genetic analysis of one sibling showed a novel frameshift mutation in exon 27 of Duchenne muscular dystrophy (DMD) gene (c.3779_3785delCTTTGGAins GG), in which 7 base pairs are deleted and two are inserted. While this predicts an amino acid substitution and premature termination (p.Thr1260Argfs*8), muscle biopsy dystrophin immunostaining instead indicates that the mutation is more likely to alter splicing. Despite relatively preserved skeletal muscular performance, both siblings developed progressive heart failure secondary to early onset DCM. In addition, their 7 year old nephew with delayed gross motor development, mild proximal muscle weakness, and markedly elevated serum creatine kinase (CK) level (> 13,000 IU/L) at 16 months was recently demonstrated to have the familial DMD mutation. Here we report a novel genotype of BMD with early onset DCM and progressive lethal heart failure during early adolescence. PMID:25537791

  14. COMMUNICATION: Alternative splicing and genomic stability

    NASA Astrophysics Data System (ADS)

    Cahill, Kevin

    2004-06-01

    Alternative splicing allows an organism to make different proteins in different cells at different times, all from the same gene. In a cell that uses alternative splicing, the total length of all the exons is much shorter than in a cell that encodes the same set of proteins without alternative splicing. This economical use of exons makes genes more stable during reproduction and development because a genome with a shorter exon length is more resistant to harmful mutations. Genomic stability may be the reason why higher vertebrates splice alternatively. For a broad class of alternatively spliced genes, a formula is given for the increase in their stability.

  15. Bioinformatic and functional optimization of antisense phosphorodiamidate morpholino oligomers (PMOs) for therapeutic modulation of RNA splicing in muscle.

    PubMed

    Popplewell, Linda J; Graham, Ian R; Malerba, Alberto; Dickson, George

    2011-01-01

    Duchenne muscular dystrophy (DMD) is caused by mutations that disrupt the reading frame of the human DMD gene. Selective removal of exons flanking an out-of-frame DMD mutation can result in an in-frame mRNA transcript that may be translated into an internally deleted, Becker muscular dystrophy (BMD)-like, but functionally active dystrophin protein with therapeutic activity. Antisense oligonucleotides (AOs) can be designed to bind to complementary sequences in the targeted mRNA and modify pre-mRNA splicing to correct the reading frame of a mutated transcript so that gene expression is restored. AO-induced exon skipping producing functional truncated dystrophin exon has been demonstrated in animal models of DMD both in vitro and in vivo, and in DMD patient cells in vitro in culture, and in DMD muscle explants. More recently, AO-mediated exon skipping has been confirmed in DMD patients in Phase I clinical trials. However, it should be noted that personalized molecular medicine may be necessary, since the various reading frame-disrupting mutations are spread across the DMD gene. The different deletions that cause DMD would require skipping of different exons, which would require the optimization and clinical trial workup of many specific AOs. This chapter describes the methodologies available for the optimization of AOs, and in particular phosphorodiamidate morpholino oligomers (PMOs), for the targeted skipping of specific exons on the DMD gene. PMID:21194027

  16. Evolution of alternative splicing after gene duplication.

    PubMed

    Su, Zhixi; Wang, Jianmin; Yu, Jun; Huang, Xiaoqiu; Gu, Xun

    2006-02-01

    Alternative splicing and gene duplication are two major sources of proteomic function diversity. Here, we study the evolutionary trend of alternative splicing after gene duplication by analyzing the alternative splicing differences between duplicate genes. We observed that duplicate genes have fewer alternative splice (AS) forms than single-copy genes, and that a negative correlation exists between the mean number of AS forms and the gene family size. Interestingly, we found that the loss of alternative splicing in duplicate genes may occur shortly after the gene duplication. These results support the subfunctionization model of alternative splicing in the early stage after gene duplication. Further analysis of the alternative splicing distribution in human duplicate pairs showed the asymmetric evolution of alternative splicing after gene duplications; i.e., the AS forms between duplicates may differ dramatically. We therefore conclude that alternative splicing and gene duplication may not evolve independently. In the early stage after gene duplication, young duplicates may take over a certain amount of protein function diversity that previously was carried out by the alternative splicing mechanism. In the late stage, the gain and loss of alternative splicing seem to be independent between duplicates. PMID:16365379

  17. The RNA Splicing Response to DNA Damage.

    PubMed

    Shkreta, Lulzim; Chabot, Benoit

    2015-01-01

    The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging. PMID:26529031

  18. The RNA Splicing Response to DNA Damage

    PubMed Central

    Shkreta, Lulzim; Chabot, Benoit

    2015-01-01

    The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging. PMID:26529031

  19. Traceless protein splicing utilizing evolved split inteins

    PubMed Central

    Lockless, Steve W.; Muir, Tom W.

    2009-01-01

    Split inteins are parasitic genetic elements frequently found inserted into reading frames of essential proteins. Their association and excision restore host protein function through a protein self-splicing reaction. They have gained an increasingly important role in the chemical modification of proteins to create cyclical, segmentally labeled, and fluorescently tagged proteins. Ideally, inteins would seamlessly splice polypeptides together with no remnant sequences and at high efficiency. Here, we describe experiments that identify the branched intermediate, a transient step in the overall splicing reaction, as a key determinant of the splicing efficiency at different splice-site junctions. To alter intein specificity, we developed a cell-based selection scheme to evolve split inteins that splice with high efficiency at different splice junctions and at higher temperatures. Mutations within these evolved inteins occur at sites distant from the active site. We present a hypothesis that a network of conserved coevolving amino acids in inteins mediates these long-range effects. PMID:19541616

  20. The genetics of splicing in neuroblastoma

    PubMed Central

    Chen, Justin; Hackett, Christopher S.; Zhang, Shile; Song, Young K.; Bell, Robert J.A.; Molinaro, Annette M.; Quigley, David A.; Balmain, Allan; Song, Jun S.; Costello, Joseph F.; Gustafson, W. Clay; Dyke, Terry Van; Kwok, Pui-Yan; Khan, Javed; Weiss, William A.

    2015-01-01

    Regulation of mRNA splicing, a critical and tightly regulated cellular function, underlies the majority of proteomic diversity, and is frequently disrupted in disease. Using an integrative genomics approach, we combined both genome and exon level transcriptome data in two somatic tissues (cerebella and peripheral ganglia) from a transgenic mouse model of neuroblastoma, a tumor that arises from peripheral neural crest. Here we describe splicing quantitative trait loci (sQTL) associated with differential splicing across the genome that we use to identify genes with previously unknown functions within the splicing pathway and to define de novo intronic splicing motifs that influence splicing from hundreds of bases away. Our results show that these splicing motifs represent sites for functional recurrent mutations and highlight novel candidate genes in human cancers, including childhood neuroblastoma. PMID:25637275

  1. Comprehensive splicing functional analysis of DNA variants of the BRCA2 gene by hybrid minigenes

    PubMed Central

    2012-01-01

    Introduction The underlying pathogenic mechanism of a large fraction of DNA variants of disease-causing genes is the disruption of the splicing process. We aimed to investigate the effect on splicing of the BRCA2 variants c.8488-1G > A (exon 20) and c.9026_9030del (exon 23), as well as 41 BRCA2 variants reported in the Breast Cancer Information Core (BIC) mutation database. Methods DNA variants were analyzed with the splicing prediction programs NNSPLICE and Human Splicing Finder. Functional analyses of candidate variants were performed by lymphocyte RT-PCR and/or hybrid minigene assays. Forty-one BIC variants of exons 19, 20, 23 and 24 were bioinformatically selected and generated by PCR-mutagenesis of the wild type minigenes. Results Lymphocyte RT-PCR of c.8488-1G > A showed intron 19 retention and a 12-nucleotide deletion in exon 20, whereas c.9026_9030del did not show any splicing anomaly. Minigene analysis of c.8488-1G > A displayed the aforementioned aberrant isoforms but also exon 20 skipping. We further evaluated the splicing outcomes of 41 variants of four BRCA2 exons by minigene analysis. Eighteen variants presented splicing aberrations. Most variants (78.9%) disrupted the natural splice sites, whereas four altered putative enhancers/silencers and had a weak effect. Fluorescent RT-PCR of minigenes accurately detected 14 RNA isoforms generated by cryptic site usage, exon skipping and intron retention events. Fourteen variants showed total splicing disruptions and were predicted to truncate or eliminate essential domains of BRCA2. Conclusions A relevant proportion of BRCA2 variants are correlated with splicing disruptions, indicating that RNA analysis is a valuable tool to assess the pathogenicity of a particular DNA change. The minigene system is a straightforward and robust approach to detect variants with an impact on splicing and contributes to a better knowledge of this gene expression step. PMID:22632462

  2. The role of splicing factors in deregulation of alternative splicing during oncogenesis and tumor progression

    PubMed Central

    Shilo, Asaf; Siegfried, Zahava; Karni, Rotem

    2015-01-01

    In past decades, cancer research has focused on genetic alterations that are detected in malignant tissues and contribute to the initiation and progression of cancer. These changes include mutations, copy number variations, and translocations. However, it is becoming increasingly clear that epigenetic changes, including alternative splicing, play a major role in cancer development and progression. There are relatively few studies on the contribution of alternative splicing and the splicing factors that regulate this process to cancer development and progression. Recently, multiple studies have revealed altered splicing patterns in cancers and several splicing factors were found to contribute to tumor development. Studies using high-throughput genomic analysis have identified mutations in components of the core splicing machinery and in splicing factors in several cancers. In this review, we will highlight new findings on the role of alternative splicing and its regulators in cancer initiation and progression, in addition to novel approaches to correct oncogenic splicing. PMID:27308389

  3. Spliced leader RNA trans-splicing discovered in copepods

    PubMed Central

    Yang, Feifei; Xu, Donghui; Zhuang, Yunyun; Yi, Xiaoyan; Huang, Yousong; Chen, Hongju; Lin, Senjie; Campbell, David A.; Sturm, Nancy R.; Liu, Guangxing; Zhang, Huan

    2015-01-01

    Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3′-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods. PMID:26621068

  4. Spliced leader RNA trans-splicing discovered in copepods

    NASA Astrophysics Data System (ADS)

    Yang, Feifei; Xu, Donghui; Zhuang, Yunyun; Yi, Xiaoyan; Huang, Yousong; Chen, Hongju; Lin, Senjie; Campbell, David A.; Sturm, Nancy R.; Liu, Guangxing; Zhang, Huan

    2015-12-01

    Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3‧-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods.

  5. Spliced leader RNA trans-splicing discovered in copepods.

    PubMed

    Yang, Feifei; Xu, Donghui; Zhuang, Yunyun; Yi, Xiaoyan; Huang, Yousong; Chen, Hongju; Lin, Senjie; Campbell, David A; Sturm, Nancy R; Liu, Guangxing; Zhang, Huan

    2015-01-01

    Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3'-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods. PMID:26621068

  6. Homologues of the Caenorhabditis elegans Fox-1 protein are neuronal splicing regulators in mammals.

    PubMed

    Underwood, Jason G; Boutz, Paul L; Dougherty, Joseph D; Stoilov, Peter; Black, Douglas L

    2005-11-01

    A vertebrate homologue of the Fox-1 protein from C. elegans was recently shown to bind to the element GCAUG and to act as an inhibitor of alternative splicing patterns in muscle. The element UGCAUG is a splicing enhancer element found downstream of numerous neuron-specific exons. We show here that mouse Fox-1 (mFox-1) and another homologue, Fox-2, are both specifically expressed in neurons in addition to muscle and heart. The mammalian Fox genes are very complex transcription units that generate transcripts from multiple promoters and with multiple internal exons whose inclusion is regulated. These genes produce a large family of proteins with variable N and C termini and internal deletions. We show that the overexpression of both Fox-1 and Fox-2 isoforms specifically activates splicing of neuronally regulated exons. This splicing activation requires UGCAUG enhancer elements. Conversely, RNA interference-mediated knockdown of Fox protein expression inhibits splicing of UGCAUG-dependent exons. These experiments show that this large family of proteins regulates splicing in the nervous system. They do this through a splicing enhancer function, in addition to their apparent negative effects on splicing in vertebrate muscle and in worms. PMID:16260614

  7. Structure of the human myelin/oligodendrocyte glycoprotein gene and multiple alternative spliced isoforms

    SciTech Connect

    Pham-Dinh, D.; Gaspera, D.B.; Dautigny, A.

    1995-09-20

    Myelin/oligodendrocyte glycoprotein (MOG), a special component of the central nervous system localization on the outermost lamellae of mature myelin, is a member of the immunoglobulin superfamily. We report here the organization of the human MOG gene, which spans approximately 17 kb, and the characterization of six MOG mRNA splicing variants. The intron/exon structure of the human MOG gene confirmed the splicing pattern, supporting the hypothesis that mRNA isoforms could arise by alternative splicing of a single gene. In addition to the eight exons coding for the major MOG isoform, the human MOG gene also contains 3` region, a previously unknown alternatively spliced coding exon, VIA. Alternative utilization of two acceptor splicing sites for exon VIII could produce two different C-termini. The nucleotide sequences presented here may be a useful tool to study further possible involvement if the MOG gene in hereditary neurological disorders. 23 refs., 5 figs.

  8. Developmental regulation of tau splicing is disrupted in stem cell-derived neurons from frontotemporal dementia patients with the 10 + 16 splice-site mutation in MAPT.

    PubMed

    Sposito, Teresa; Preza, Elisavet; Mahoney, Colin J; Setó-Salvia, Núria; Ryan, Natalie S; Morris, Huw R; Arber, Charles; Devine, Michael J; Houlden, Henry; Warner, Thomas T; Bushell, Trevor J; Zagnoni, Michele; Kunath, Tilo; Livesey, Frederick J; Fox, Nick C; Rossor, Martin N; Hardy, John; Wray, Selina

    2015-09-15

    The alternative splicing of the tau gene, MAPT, generates six protein isoforms in the adult human central nervous system (CNS). Tau splicing is developmentally regulated and dysregulated in disease. Mutations in MAPT that alter tau splicing cause frontotemporal dementia (FTD) with tau pathology, providing evidence for a causal link between altered tau splicing and disease. The use of induced pluripotent stem cell (iPSC)-derived neurons has revolutionized the way we model neurological disease in vitro. However, as most tau mutations are located within or around the alternatively spliced exon 10, it is important that iPSC-neurons splice tau appropriately in order to be used as disease models. To address this issue, we analyzed the expression and splicing of tau in iPSC-derived cortical neurons from control patients and FTD patients with the 10 + 16 intronic mutation in MAPT. We show that control neurons only express the fetal tau isoform (0N3R), even at extended time points of 100 days in vitro. Neurons from FTD patients with the 10 + 16 mutation in MAPT express both 0N3R and 0N4R tau isoforms, demonstrating that this mutation overrides the developmental regulation of exon 10 inclusion in our in vitro model. Further, at extended time points of 365 days in vitro, we observe a switch in tau splicing to include six tau isoforms as seen in the adult human CNS. Our results demonstrate the importance of neuronal maturity for use in in vitro modeling and provide a system that will be important for understanding the functional consequences of altered tau splicing. PMID:26136155

  9. Developmental regulation of tau splicing is disrupted in stem cell-derived neurons from frontotemporal dementia patients with the 10 + 16 splice-site mutation in MAPT

    PubMed Central

    Sposito, Teresa; Preza, Elisavet; Mahoney, Colin J.; Setó-Salvia, Núria; Ryan, Natalie S.; Morris, Huw R.; Arber, Charles; Devine, Michael J.; Houlden, Henry; Warner, Thomas T.; Bushell, Trevor J.; Zagnoni, Michele; Kunath, Tilo; Livesey, Frederick J.; Fox, Nick C.; Rossor, Martin N.; Hardy, John; Wray, Selina

    2015-01-01

    The alternative splicing of the tau gene, MAPT, generates six protein isoforms in the adult human central nervous system (CNS). Tau splicing is developmentally regulated and dysregulated in disease. Mutations in MAPT that alter tau splicing cause frontotemporal dementia (FTD) with tau pathology, providing evidence for a causal link between altered tau splicing and disease. The use of induced pluripotent stem cell (iPSC)-derived neurons has revolutionized the way we model neurological disease in vitro. However, as most tau mutations are located within or around the alternatively spliced exon 10, it is important that iPSC–neurons splice tau appropriately in order to be used as disease models. To address this issue, we analyzed the expression and splicing of tau in iPSC-derived cortical neurons from control patients and FTD patients with the 10 + 16 intronic mutation in MAPT. We show that control neurons only express the fetal tau isoform (0N3R), even at extended time points of 100 days in vitro. Neurons from FTD patients with the 10 + 16 mutation in MAPT express both 0N3R and 0N4R tau isoforms, demonstrating that this mutation overrides the developmental regulation of exon 10 inclusion in our in vitro model. Further, at extended time points of 365 days in vitro, we observe a switch in tau splicing to include six tau isoforms as seen in the adult human CNS. Our results demonstrate the importance of neuronal maturity for use in in vitro modeling and provide a system that will be important for understanding the functional consequences of altered tau splicing. PMID:26136155

  10. Hallmarks of alternative splicing in cancer.

    PubMed

    Oltean, S; Bates, D O

    2014-11-13

    The immense majority of genes are alternatively spliced and there are many isoforms specifically associated with cancer progression and metastasis. The splicing pattern of specific isoforms of numerous genes is altered as cells move through the oncogenic process of gaining proliferative capacity, acquiring angiogenic, invasive, antiapoptotic and survival properties, becoming free from growth factor dependence and growth suppression, altering their metabolism to cope with hypoxia, enabling them to acquire mechanisms of immune escape, and as they move through the epithelial-mesenchymal and mesenchymal-epithelial transitions and metastasis. Each of the 'hallmarks of cancer' is associated with a switch in splicing, towards a more aggressive invasive cancer phenotype. The choice of isoforms is regulated by several factors (signaling molecules, kinases, splicing factors) currently being identified systematically by a number of high-throughput, independent and unbiased methodologies. Splicing factors are de-regulated in cancer, and in some cases are themselves oncogenes or pseudo-oncogenes and can contribute to positive feedback loops driving cancer progression. Tumour progression may therefore be associated with a coordinated splicing control, meaning that there is the potential for a relatively small number of splice factors or their regulators to drive multiple oncogenic processes. The understanding of how splicing contributes to the various phenotypic traits acquired by tumours as they progress and metastasise, and in particular how alternative splicing is coordinated, can and is leading to the development of a new class of anticancer therapeutics-the alternative-splicing inhibitors. PMID:24336324

  11. 46 CFR 111.60-19 - Cable splices.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Cable splices. 111.60-19 Section 111.60-19 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL... with section 25.11 of IEEE 45-2002 (incorporated by reference; see 46 CFR 110.10-1)....

  12. Our trails and trials in the subsarcolemmal cytoskeleton network and muscular dystrophy researches in the dystrophin era

    PubMed Central

    OZAWA, Eijiro

    2010-01-01

    In 1987, about 150 years after the discovery of Duchenne muscular dystrophy (DMD), its responsible gene, the dystrophin gene, was cloned by Kunkel. This was a new substance. During these 20 odd years after the cloning, our understanding on dystrophin as a component of the subsarcolemmal cytoskeleton networks and on the pathomechanisms of and experimental therapeutics for DMD has been greatly enhanced. During this paradigm change, I was fortunately able to work as an active researcher on its frontiers for 12 years. After we discovered that dystrophin is located on the cell membrane in 1988, we studied the architecture of dystrophin and dystrophin-associated proteins (DAPs) complex in order to investigate the function of dystrophin and pathomechanism of DMD. During the conduct of these studies, we came to consider that the dystrophin–DAP complex serves to transmembranously connect the subsarcolemmal cytoskeleton networks and basal lamina to protect the lipid bilayer. It then became our working hypothesis that injury of the lipid bilayer upon muscle contraction is the cause of DMD. During this process, we predicted that subunits of the sarcoglycan (SG) complex are responsible for respective types of DMD-like muscular dystrophy with autosomal recessive inheritance. Our prediction was confirmed to be true by many researchers including ourselves. In this review, I will try to explain what we observed and how we considered concerning the architecture and function of the dystrophin–DAP complex, and the pathomechanisms of DMD and related muscular dystrophies. PMID:20948175

  13. Phosphorylation within the cysteine-rich region of dystrophin enhances its association with β-dystroglycan and identifies a potential novel therapeutic target for skeletal muscle wasting

    PubMed Central

    Swiderski, Kristy; Shaffer, Scott A.; Gallis, Byron; Odom, Guy L.; Arnett, Andrea L.; Scott Edgar, J.; Baum, Dale M.; Chee, Annabel; Naim, Timur; Gregorevic, Paul; Murphy, Kate T.; Moody, James; Goodlett, David R.; Lynch, Gordon S.; Chamberlain, Jeffrey S.

    2014-01-01

    Mutations in dystrophin lead to Duchenne muscular dystrophy, which is among the most common human genetic disorders. Dystrophin nucleates assembly of the dystrophin–glycoprotein complex (DGC), and a defective DGC disrupts an essential link between the intracellular cytoskeleton and the basal lamina, leading to progressive muscle wasting. In vitro studies have suggested that dystrophin phosphorylation may affect interactions with actin or syntrophin, yet whether this occurs in vivo or affects protein function remains unknown. Utilizing nanoflow liquid chromatography mass spectrometry, we identified 18 phosphorylated residues within endogenous dystrophin. Mutagenesis revealed that phosphorylation at S3059 enhances the dystrophin–dystroglycan interaction and 3D modeling utilizing the Rosetta software program provided a structural model for how phosphorylation enhances this interaction. These findings demonstrate that phosphorylation is a key mechanism regulating the interaction between dystrophin and the DGC and reveal that posttranslational modification of a single amino acid directly modulates the function of dystrophin. PMID:25082828

  14. The mammalian homolog of suppressor-of-white-apricot regulates alternative mRNA splicing of CD45 exon 4 and fibronectin IIICS.

    PubMed

    Sarkissian, M; Winne, A; Lafyatis, R

    1996-12-01

    We have previously described human (HsSWAP) and mouse (MmSWAP) homologs to the Drosophila alternative splicing regulator suppressor-of-white-apricot (su(wa) or DmSWAP). DmSWAP was formally defined as an alternative splicing regulator by studies showing that it autoregulates splicing of its own pre-mRNA. We report here that mammalian SWAP regulates its own splicing, and also the splicing of fibronectin and CD45. Using an in vivo system of cell transfection, mammalian SWAP regulated 5' splice site selection in splicing of its own second intron. SWAP enhanced splicing to the distal 5' splice site, whereas the SR protein ASF/SF2 enhanced splicing to the proximal site. SWAP also regulated alternative splicing of the fibronectin IIICS region by promoting exclusion of the entire IIICS region. In contrast, ASF/SF2 stimulated inclusion of the entire IIICS region. Finally, SWAP regulated splicing of CD45 exon 4, promoting exclusion of this exon, an effect also seen with ASF/SF2. Experiments using SWAP deletion mutants showed that splicing regulation of the fibronectin IIICS region and CD45 exon 4 requires a region including a carboxyl-terminal arginine/serine (R/S)-rich motif. Since R/S motifs of various splicing proteins have been shown to interact with each other, these results suggest that the R/S motif in SWAP may regulate splicing, at least in part, through interactions with other R/S containing splicing factors. PMID:8940107

  15. Safely splicing glass optical fibers

    NASA Technical Reports Server (NTRS)

    Korbelak, K.

    1980-01-01

    Field-repair technique fuses glass fibers in flammable environment. Apparatus consists of v-groove vacuum chucks on manipulators, high-voltage dc power supply and tungsten electrodes, microscope to observe joint alignment and fusion, means of test transmission through joint. Apparatus is enclosed in gas tight bos filled with inert gas during fusion. About 2 feet of fiber end are necessary for splicing.

  16. Functional consequences of developmentally regulated alternative splicing

    PubMed Central

    Kalsotra, Auinash; Cooper, Thomas A.

    2012-01-01

    Genome-wide analyses of metazoan transcriptomes have revealed an unexpected level of mRNA diversity that is generated by alternative splicing. Recently, regulatory networks have been identified through which splicing promotes dynamic remodeling of the transcriptome to promote physiological changes, which involve robust and coordinated alternative splicing transitions. The regulation of splicing in yeast, worms, flies and vertebrates affects a variety of biological processes. The functional classes of genes that are regulated by alternative splicing include both those with widespread homeostatic activities and genes with cell-type-specific functions. Alternative splicing can drive determinative physiological change or can have a permissive role by providing mRNA variability that is utilized by other regulatory mechanisms. PMID:21921927

  17. Investigating alternative RNA splicing in Xenopus.

    PubMed

    Mereau, Agnès; Hardy, Serge

    2012-01-01

    Alternative splicing, the process by which distinct mature mRNAs can be produced from a single primary transcript, is a key mechanism to increase the organism complexity. The generation of alternative splicing pattern is a means to expand the proteome diversity and also to control gene expression through the regulation of mRNA abundance. Alternative splicing is therefore particularly prevalent during development and accordingly numerous splicing events are regulated in a tissue or temporal manner. To study the roles of alternative splicing during developmental processes and decipher the molecular mechanisms that underlie temporal and spatial regulation, it is important to develop in vivo whole animal studies. In this chapter, we present the advantages of using the amphibian Xenopus as a fully in vivo model to study alternative splicing and we describe the experimental procedures that can be used with Xenopus laevis embryos and oocytes to define the cis-regulatory elements and identify the associated trans-acting factors. PMID:22956098

  18. Splicing tolerances of weakly coupled few-mode fibers for mode-division-multiplexed transmission using sparse MIMO equalizers

    NASA Astrophysics Data System (ADS)

    Han, Jiawei

    2016-01-01

    For one-polarization mode-division-multiplexed (MDM) system using sparse 2 × 2 MIMO equalizers over twofold spatially degenerate (TSD) LP modes, we numerically investigate the discrete modal crosstalk (XT) behaviors due to splices in weakly coupled few-mode fibers. Its impacts on transmission distance of uncoupled MDM link are comparably analyzed by evaluating the extreme cases relevant to the required splicing tolerances. Under different splicing conditions, the complexity of 2 × 2 MIMO equalizers is assessed by intra-modal relative group delay (RGD) emulation. Simulation results show that, by specifically controlling the lateral offsets at splicing points, an intra-modal RGD reduction of TSD LP11 modes can be obtained, benefiting the tap number decrease of the corresponding 2 × 2 MIMO equalizer. Besides, the propagation of TSD LP21 modes is verified to possess intra-modal splice-XT-immune property, indicating that the relevant intra-modal RGD will not be affected by splices.

  19. Relatively low proportion of dystrophin gene deletions in Israeli Duchenne and Becker muscular dystrophy patients.

    PubMed

    Shomrat, R; Gluck, E; Legum, C; Shiloh, Y

    1994-02-15

    Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the X-linked dystrophin gene. The most common mutations in western populations are deletions that are spread non-randomly throughout the gene. Molecular analysis of the dystrophin gene structure by hybridization of the full length cDNA to Southern blots and by PCR in 62 unrelated Israeli male DMD/BMD patients showed deletions in 23 (37%). This proportion is significantly lower than that found in European and North American populations (55-65%). Seventy-eight percent of the deletions were confined to exons 44-52, half of these to exons 44-45, and the remaining 22% to exons 1 and 19. There was no correlation between the size of the deletion and the severity of the disease. All the deletions causing frameshift resulted in the DMD phenotypes. PMID:8160727

  20. Finding the sweet spot: Assembly and Glycosylation of the Dystrophin-Associated Glycoprotein Complex

    PubMed Central

    Townsend, DeWayne

    2014-01-01

    The dystrophin-associated glycoprotein complex (DGC) is a collection of glycoproteins that are essential for the normal function of striated muscle and many other tissues. Recent genetic studies have implicated the components of this complex in over a dozen forms of muscular dystrophy. Furthermore, disruption of the DGC has been implicated in many forms of acquired disease. This review aims to summarize the current state of knowledge regarding the processing and assembly of dystrophin associated proteins with a focus primarily on the dystroglycan heterodimer and the sarcoglycan complex. These proteins form the transmembrane portion of the DGC and undergo a complex multi-step processing with proteolytic cleavage, differential assembly, and both N- and O-glycosylation. The enzymes responsible for this processing and a model describing the sequence and subcellular localization of these events are discussed. PMID:25125182

  1. Dystrophin Gene Replacement and Gene Repair Therapy for Duchenne Muscular Dystrophy in 2016: An Interview.

    PubMed

    Duan, Dongsheng

    2016-03-01

    After years of relentless efforts, gene therapy has now begun to deliver its therapeutic promise in several diseases. A number of gene therapy products have received regulatory approval in Europe and Asia. Duchenne muscular dystrophy (DMD) is an X-linked inherited lethal muscle disease. It is caused by mutations in the dystrophin gene. Replacing and/or repairing the mutated dystrophin gene holds great promises to treated DMD at the genetic level. Last several years have evidenced significant developments in preclinical experimentations in murine and canine models of DMD. There has been a strong interest in moving these promising findings to clinical trials. In light of rapid progress in this field, the Parent Project Muscular Dystrophy (PPMD) recently interviewed me on the current status of DMD gene therapy and readiness for clinical trials. Here I summarized the interview with PPMD. PMID:27003751

  2. Relatively low proportion of dystrophin gene deletions in Israeili Duchenne and Becker muscular dystrophy patients

    SciTech Connect

    Shomrat, R.; Gluck, E.; Legum, C.; Shiloh, Y.

    1994-02-15

    Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the X-linked dystrophin gene. The most common mutations in western populations are deletions that are spread non-randomly throughout the gene. Molecular analysis of the dystrophin gene structure by hybridization of the full length cDNA to Southern blots and by PCR in 62 unrelated Israeli male DMD/BMD patients showed deletions in 23 (37%). This proportion is significantly lower than that found in European and North American populations (55-65%). Seventy-eight percent of the deletions were confined to exons 44-52, half of these exons 44-45, and the remaining 22% to exons 1 and 19. There was no correlation between the size of the deletion and the severity of the disease. All the deletions causing frameshift resulted in the DMD phenotypes. 43 refs., 1 fig., 1 tab.

  3. Restoration of half the normal dystrophin sequence in a double-deletion Duchenne muscular dystrophy family

    SciTech Connect

    Hoop, R.C.; Schwartz, L.S.; Hoffman, E.P.; Russo, L.S.; Riconda, D.L.

    1994-02-01

    Two male cousins with Duchenne muscular dystrophy were found to have different maternal dystrophin gene haplotypes and different deletion mutations. One propositus showed two noncontiguous deletions-one in the 5{prime}, proximal deletional hotspot region, and the other in the 3{prime}, more distal deletional hotspot region. The second propositus showed only the 5{prime} deletion. Using multiple fluorescent exon dosage and fluorescent multiplex CA repeat linkage analyses, the authors show that the mother of each propositus carries both deletions on the same grandmaternal X chromosome. This paradox is explained by a single recombinational event between the 2 deleted regions of one of the carrier`s dystrophin genes, giving rise to a son with a partially {open_quotes}repaired{close_quotes} gene retaining only the 5{prime} deletion. 20 refs., 4 figs.

  4. Prediction and Quantification of Splice Events from RNA-Seq Data

    PubMed Central

    Goldstein, Leonard D.; Cao, Yi; Pau, Gregoire; Lawrence, Michael; Wu, Thomas D.; Seshagiri, Somasekar; Gentleman, Robert

    2016-01-01

    Analysis of splice variants from short read RNA-seq data remains a challenging problem. Here we present a novel method for the genome-guided prediction and quantification of splice events from RNA-seq data, which enables the analysis of unannotated and complex splice events. Splice junctions and exons are predicted from reads mapped to a reference genome and are assembled into a genome-wide splice graph. Splice events are identified recursively from the graph and are quantified locally based on reads extending across the start or end of each splice variant. We assess prediction accuracy based on simulated and real RNA-seq data, and illustrate how different read aligners (GSNAP, HISAT2, STAR, TopHat2) affect prediction results. We validate our approach for quantification based on simulated data, and compare local estimates of relative splice variant usage with those from other methods (MISO, Cufflinks) based on simulated and real RNA-seq data. In a proof-of-concept study of splice variants in 16 normal human tissues (Illumina Body Map 2.0) we identify 249 internal exons that belong to known genes but are not related to annotated exons. Using independent RNA samples from 14 matched normal human tissues, we validate 9/9 of these exons by RT-PCR and 216/249 by paired-end RNA-seq (2 x 250 bp). These results indicate that de novo prediction of splice variants remains beneficial even in well-studied systems. An implementation of our method is freely available as an R/Bioconductor package SGSeq. PMID:27218464

  5. Orphan drug development in muscular dystrophy: update on two large clinical trials of dystrophin rescue therapies.

    PubMed

    Hoffman, Eric P; Connor, Edward M

    2013-11-01

    Duchenne muscular dystrophy is a relatively common 'rare disorder,' with an incidence of about 1/5,000 males worldwide. The responsible gene and deficient protein (dystrophin) were identified in 1987, an early success of human molecular genetics and emerging genome projects. A rational approach to therapeutics is to replace dystrophin in patient muscle, thus addressing the primary biochemical defect. Fast forward 25 years, and two phase 2b/3 trials have been carried out with agents designed to induce de novo dystrophin production in DMD patient's muscle; ataluren (stop codon read through) with 174 patients, and drisapersen (exon skipping) with 186 patients. Both used a six minute walk test as the primary outcome measure. Neither drisapersen nor high dose ataluren showed any significant improvement in this outcome, whereas low dose ataluren is reported to show some possible improvement. Experience with ataluren and drisapersen has been disappointing and this is a good time to ask: What can we learn from these programs and how can this inform further drug development in DMD? At the times these two trials were started, there was a lack of existing data and infrastructure regarding both clinical and biochemical outcome measures. The recent publications of more extensive natural history data in multiple DMD cohorts, and ongoing efforts to define reliable and sensitive dystrophin assays are important. If the drisapersen and ataluren programs were instead begun today, new progress in biochemical and clinical endpoints may have triggered a re-design, with better de-risking in phase 2 studies prior to resource-intensive phase 3 trials. PMID:24229740

  6. Ex Vivo Stretch Reveals Altered Mechanical Properties of Isolated Dystrophin-Deficient Hearts

    PubMed Central

    Barnabei, Matthew S.; Metzger, Joseph M.

    2012-01-01

    Duchenne muscular dystrophy (DMD) is a progressive and fatal disease of muscle wasting caused by loss of the cytoskeletal protein dystrophin. In the heart, DMD results in progressive cardiomyopathy and dilation of the left ventricle through mechanisms that are not fully understood. Previous reports have shown that loss of dystrophin causes sarcolemmal instability and reduced mechanical compliance of isolated cardiac myocytes. To expand upon these findings, here we have subjected the left ventricles of dystrophin-deficient mdx hearts to mechanical stretch. Unexpectedly, isolated mdx hearts showed increased left ventricular (LV) compliance compared to controls during stretch as LV volume was increased above normal end diastolic volume. During LV chamber distention, sarcomere lengths increased similarly in mdx and WT hearts despite greater excursions in volume of mdx hearts. This suggests that the mechanical properties of the intact heart cannot be modeled as a simple extrapolation of findings in single cardiac myocytes. To explain these findings, a model is proposed in which disruption of the dystrophin-glycoprotein complex perturbs cell-extracellular matrix contacts and promotes the apparent slippage of myocytes past each other during LV distension. In comparison, similar increases in LV compliance were obtained in isolated hearts from β-sarcoglycan-null and laminin-α2 mutant mice, but not in dysferlin-null mice, suggesting that increased whole-organ compliance in mdx mice is a specific effect of disrupted cell-extracellular matrix contacts and not a general consequence of cardiomyopathy via membrane defect processes. Collectively, these findings suggest a novel and cell-death independent mechanism for the progressive pathological LV dilation that occurs in DMD. PMID:22427904

  7. Intellectual Ability in the Duchenne Muscular Dystrophy and Dystrophin Gene Mutation Location

    PubMed Central

    Milic Rasic, V; Vojinovic, D; Pesovic, J; Mijalkovic, G; Lukic, V; Mladenovic, J; Kosac, A; Novakovic, I; Maksimovic, N; Romac, S; Todorovic, S; Savic Pavicevic, D

    2014-01-01

    Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy during childhood. Mutations in dystrophin (DMD) gene are also recognized as a cause of cognitive impairment. We aimed to determine the association between intelligence level and mutation location in DMD genes in Serbian patients with DMD. Forty-one male patients with DMD, aged 3 to 16 years, were recruited at the Clinic for Neurology and Psychiatry for Children and Youth in Belgrade, Serbia. All patients had defined DMD gene deletions or duplications [multiplex ligation-dependent probe amplification (MLPA), polymerase chain reaction (PCR)] and cognitive status assessment (Wechsler Intelligence Scale for Children, Brunet-Lezine scale, Vineland-Doll scale). In 37 patients with an estimated full scale intelligence quotient (FSIQ), six (16.22%) had borderline intelligence (70dystrophin isoforms and when mutations in the 5′-untranslated region (5′UTR) of Dp140 (exons 45–50) were assigned to affect only Dp427 and Dp260. Mutations affecting Dp140 and Dp71/Dp40 have been associated with more frequent and more severe cognitive impairment. Finally, the same classification of mutations explained the greater proportion of FSIQ variability associated with cumulative loss of dystrophin isoforms. In conclusion, cumulative loss of dystrophin isoforms increases the risk of intellectual impairment in DMD and characterizing the genotype can define necessity of early cognitive interventions in DMD patients. PMID:25937795

  8. Intellectual ability in the duchenne muscular dystrophy and dystrophin gene mutation location.

    PubMed

    Milic Rasic, V; Vojinovic, D; Pesovic, J; Mijalkovic, G; Lukic, V; Mladenovic, J; Kosac, A; Novakovic, I; Maksimovic, N; Romac, S; Todorovic, S; Savic Pavicevic, D

    2014-12-01

    Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy during childhood. Mutations in dystrophin (DMD) gene are also recognized as a cause of cognitive impairment. We aimed to determine the association between intelligence level and mutation location in DMD genes in Serbian patients with DMD. Forty-one male patients with DMD, aged 3 to 16 years, were recruited at the Clinic for Neurology and Psychiatry for Children and Youth in Belgrade, Serbia. All patients had defined DMD gene deletions or duplications [multiplex ligation-dependent probe amplification (MLPA), polymerase chain reaction (PCR)] and cognitive status assessment (Wechsler Intelligence Scale for Children, Brunet-Lezine scale, Vineland-Doll scale). In 37 patients with an estimated full scale intelligence quotient (FSIQ), six (16.22%) had borderline intelligence (70dystrophin isoforms and when mutations in the 5'-untranslated region (5'UTR) of Dp140 (exons 45-50) were assigned to affect only Dp427 and Dp260. Mutations affecting Dp140 and Dp71/Dp40 have been associated with more frequent and more severe cognitive impairment. Finally, the same classification of mutations explained the greater proportion of FSIQ variability associated with cumulative loss of dystrophin isoforms. In conclusion, cumulative loss of dystrophin isoforms increases the risk of intellectual impairment in DMD and characterizing the genotype can define necessity of early cognitive interventions in DMD patients. PMID:25937795

  9. Recursive splicing in long vertebrate genes

    PubMed Central

    Blazquez, Lorea; Faro, Ana; Haberman, Nejc; Briese, Michael; Trabzuni, Daniah; Ryten, Mina; Weale, Michael E; Hardy, John; Modic, Miha; Curk, Tomaž; Wilson, Stephen W; Plagnol, Vincent; Ule, Jernej

    2015-01-01

    It is generally believed that splicing removes introns as single units from pre-mRNA transcripts. However, some long D. melanogaster introns contain a cryptic site, called a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing1,2. The extent to which recursive splicing occurs in other species and its mechanistic basis remain unclear. Here we identify highly conserved RS-sites in genes expressed in the mammalian brain that encode proteins functioning in neuronal development. Moreover, the RS-sites are found in some of the longest introns across vertebrates. We find that vertebrate recursive splicing requires initial definition of a “RS-exon” that follows the RS-site. The RS-exon is then excluded from the dominant mRNA isoform due to competition with a reconstituted 5′ splice site formed at the RS-site after the first splicing step. Conversely, the RS-exon is included when preceded by cryptic exons or promoters that are prevalent in long introns, but which fail to reconstitute an efficient 5′ splice site. Most RS-exons contain a premature stop codon such that their inclusion may decrease mRNA stability. Thus, by establishing a binary splicing switch, RS-sites demarcate different mRNA isoforms emerging from long genes by coupling inclusion of cryptic elements with RS-exons. PMID:25970246

  10. Recursive splicing in long vertebrate genes.

    PubMed

    Sibley, Christopher R; Emmett, Warren; Blazquez, Lorea; Faro, Ana; Haberman, Nejc; Briese, Michael; Trabzuni, Daniah; Ryten, Mina; Weale, Michael E; Hardy, John; Modic, Miha; Curk, Tomaž; Wilson, Stephen W; Plagnol, Vincent; Ule, Jernej

    2015-05-21

    It is generally believed that splicing removes introns as single units from precursor messenger RNA transcripts. However, some long Drosophila melanogaster introns contain a cryptic site, known as a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing. The extent to which recursive splicing occurs in other species and its mechanistic basis have not been examined. Here we identify highly conserved RS-sites in genes expressed in the mammalian brain that encode proteins functioning in neuronal development. Moreover, the RS-sites are found in some of the longest introns across vertebrates. We find that vertebrate recursive splicing requires initial definition of an 'RS-exon' that follows the RS-site. The RS-exon is then excluded from the dominant mRNA isoform owing to competition with a reconstituted 5' splice site formed at the RS-site after the first splicing step. Conversely, the RS-exon is included when preceded by cryptic promoters or exons that fail to reconstitute an efficient 5' splice site. Most RS-exons contain a premature stop codon such that their inclusion can decrease mRNA stability. Thus, by establishing a binary splicing switch, RS-sites demarcate different mRNA isoforms emerging from long genes by coupling cryptic elements with inclusion of RS-exons. PMID:25970246

  11. Regulation of Splicing Factors by Alternative Splicing and NMD Is Conserved between Kingdoms Yet Evolutionarily Flexible

    PubMed Central

    Lareau, Liana F.; Brenner, Steven E.

    2015-01-01

    Ultraconserved elements, unusually long regions of perfect sequence identity, are found in genes encoding numerous RNA-binding proteins including arginine-serine rich (SR) splicing factors. Expression of these genes is regulated via alternative splicing of the ultraconserved regions to yield mRNAs that are degraded by nonsense-mediated mRNA decay (NMD), a process termed unproductive splicing (Lareau et al. 2007; Ni et al. 2007). As all human SR genes are affected by alternative splicing and NMD, one might expect this regulation to have originated in an early SR gene and persisted as duplications expanded the SR family. But in fact, unproductive splicing of most human SR genes arose independently (Lareau et al. 2007). This paradox led us to investigate the origin and proliferation of unproductive splicing in SR genes. We demonstrate that unproductive splicing of the splicing factor SRSF5 (SRp40) is conserved among all animals and even observed in fungi; this is a rare example of alternative splicing conserved between kingdoms, yet its effect is to trigger mRNA degradation. As the gene duplicated, the ancient unproductive splicing was lost in paralogs, and distinct unproductive splicing evolved rapidly and repeatedly to take its place. SR genes have consistently employed unproductive splicing, and while it is exceptionally conserved in some of these genes, turnover in specific events among paralogs shows flexible means to the same regulatory end. PMID:25576366

  12. CRISPR-mediated Genome Editing Restores Dystrophin Expression and Function in mdx Mice.

    PubMed

    Xu, Li; Park, Ki Ho; Zhao, Lixia; Xu, Jing; El Refaey, Mona; Gao, Yandi; Zhu, Hua; Ma, Jianjie; Han, Renzhi

    2016-03-01

    Duchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by genetic mutations that lead to the disruption of dystrophin in muscle fibers. There is no curative treatment for this devastating disease. Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) has emerged as a powerful tool for genetic manipulation and potential therapy. Here we demonstrate that CRIPSR-mediated genome editing efficiently excised a 23-kb genomic region on the X-chromosome covering the mutant exon 23 in a mouse model of DMD, and restored dystrophin expression and the dystrophin-glycoprotein complex at the sarcolemma of skeletal muscles in live mdx mice. Electroporation-mediated transfection of the Cas9/gRNA constructs in the skeletal muscles of mdx mice normalized the calcium sparks in response to osmotic shock. Adenovirus-mediated transduction of Cas9/gRNA greatly reduced the Evans blue dye uptake of skeletal muscles at rest and after downhill treadmill running. This study provides proof evidence for permanent gene correction in DMD. PMID:26449883

  13. Muscle inactivation of mTOR causes metabolic and dystrophin defects leading to severe myopathy

    PubMed Central

    Risson, Valérie; Mazelin, Laetitia; Roceri, Mila; Sanchez, Hervé; Moncollin, Vincent; Corneloup, Claudine; Richard-Bulteau, Hélène; Vignaud, Alban; Baas, Dominique; Defour, Aurélia; Freyssenet, Damien; Tanti, Jean-François; Le-Marchand-Brustel, Yannick; Ferrier, Bernard; Conjard-Duplany, Agnès; Romanino, Klaas; Bauché, Stéphanie; Hantaï, Daniel; Mueller, Matthias; Kozma, Sara C.; Thomas, George; Rüegg, Markus A.; Ferry, Arnaud; Pende, Mario; Bigard, Xavier; Koulmann, Nathalie

    2009-01-01

    Mammalian target of rapamycin (mTOR) is a key regulator of cell growth that associates with raptor and rictor to form the mTOR complex 1 (mTORC1) and mTORC2, respectively. Raptor is required for oxidative muscle integrity, whereas rictor is dispensable. In this study, we show that muscle-specific inactivation of mTOR leads to severe myopathy, resulting in premature death. mTOR-deficient muscles display metabolic changes similar to those observed in muscles lacking raptor, including impaired oxidative metabolism, altered mitochondrial regulation, and glycogen accumulation associated with protein kinase B/Akt hyperactivation. In addition, mTOR-deficient muscles exhibit increased basal glucose uptake, whereas whole body glucose homeostasis is essentially maintained. Importantly, loss of mTOR exacerbates the myopathic features in both slow oxidative and fast glycolytic muscles. Moreover, mTOR but not raptor and rictor deficiency leads to reduced muscle dystrophin content. We provide evidence that mTOR controls dystrophin transcription in a cell-autonomous, rapamycin-resistant, and kinase-independent manner. Collectively, our results demonstrate that mTOR acts mainly via mTORC1, whereas regulation of dystrophin is raptor and rictor independent. PMID:20008564

  14. Spatio-temporal regulations and functions of neuronal alternative RNA splicing in developing and adult brains.

    PubMed

    Iijima, Takatoshi; Hidaka, Chiharu; Iijima, Yoko

    2016-08-01

    Alternative pre-mRNA splicing is a fundamental mechanism that generates molecular diversity from a single gene. In the central nervous system (CNS), key neural developmental steps are thought to be controlled by alternative splicing decisions, including the molecular diversity underlying synaptic wiring, plasticity, and remodeling. Significant progress has been made in understanding the molecular mechanisms and functions of alternative pre-mRNA splicing in neurons through studies in invertebrate systems; however, recent studies have begun to uncover the potential role of neuronal alternative splicing in the mammalian CNS. This article provides an overview of recent findings regarding the regulation and function of neuronal alternative splicing. In particular, we focus on the spatio-temporal regulation of neurexin, a synaptic adhesion molecule, by neuronal cell type-specific factors and neuronal activity, which are thought to be especially important for characterizing neural development and function within the mammalian CNS. Notably, there is increasing evidence that implicates the dysregulation of neuronal splicing events in several neurological disorders. Therefore, understanding the detailed mechanisms of neuronal alternative splicing in the mammalian CNS may provide plausible treatment strategies for these diseases. PMID:26853282

  15. 49 CFR 234.241 - Protection of insulated wire; splice in underground wire.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... underground wire. 234.241 Section 234.241 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION, DEPARTMENT OF TRANSPORTATION GRADE CROSSING SIGNAL SYSTEM SAFETY... of insulated wire; splice in underground wire. Insulated wire shall be protected from...

  16. 49 CFR 234.241 - Protection of insulated wire; splice in underground wire.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... underground wire. 234.241 Section 234.241 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION, DEPARTMENT OF TRANSPORTATION GRADE CROSSING SIGNAL SYSTEM SAFETY... of insulated wire; splice in underground wire. Insulated wire shall be protected from...

  17. Immobilization and therapeutic passive stretching generate thickening and increase the expression of laminin and dystrophin in skeletal muscle

    PubMed Central

    Cação-Benedini, L.O.; Ribeiro, P.G.; Prado, C.M.; Chesca, D.L.; Mattiello-Sverzut, A.C.

    2014-01-01

    Extracellular matrix and costamere proteins transmit the concentric, isometric, and eccentric forces produced by active muscle contraction. The expression of these proteins after application of passive tension stimuli to muscle remains unknown. This study investigated the expression of laminin and dystrophin in the soleus muscle of rats immobilized with the right ankle in plantar flexion for 10 days and subsequent remobilization, either by isolated free movement in a cage or associated with passive stretching for up to 10 days. The intensity of the macrophage response was also evaluated. One hundred and twenty-eight female Wistar rats were divided into 8 groups: free for 10 days; immobilized for 10 days; immobilized/free for 1, 3, or 10 days; or immobilized/stretched/free for 1, 3, or 10 days. After the experimental procedures, muscle tissue was processed for immunofluorescence (dystrophin/laminin/CD68) and Western blot analysis (dystrophin/laminin). Immobilization increased the expression of dystrophin and laminin but did not alter the number of macrophages in the muscle. In the stretched muscle groups, there was an increase in dystrophin and the number of macrophages after 3 days compared with the other groups; dystrophin showed a discontinuous labeling pattern, and laminin was found in the intracellular space. The amount of laminin was increased in the muscles treated by immobilization followed by free movement for 10 days. In the initial stages of postimmobilization (1 and 3 days), an exacerbated macrophage response and an increase of dystrophin suggested that the therapeutic stretching technique induced additional stress in the muscle fibers and costameres. PMID:24820070

  18. SplicingTypesAnno: annotating and quantifying alternative splicing events for RNA-Seq data.

    PubMed

    Sun, Xiaoyong; Zuo, Fenghua; Ru, Yuanbin; Guo, Jiqiang; Yan, Xiaoyan; Sablok, Gaurav

    2015-04-01

    Alternative splicing plays a key role in the regulation of the central dogma. Four major types of alternative splicing have been classified as intron retention, exon skipping, alternative 5 splice sites or alternative donor sites, and alternative 3 splice sites or alternative acceptor sites. A few algorithms have been developed to detect splice junctions from RNA-Seq reads. However, there are few tools targeting at the major alternative splicing types at the exon/intron level. This type of analysis may reveal subtle, yet important events of alternative splicing, and thus help gain deeper understanding of the mechanism of alternative splicing. This paper describes a user-friendly R package, extracting, annotating and analyzing alternative splicing types for sequence alignment files from RNA-Seq. SplicingTypesAnno can: (1) provide annotation for major alternative splicing at exon/intron level. By comparing the annotation from GTF/GFF file, it identifies the novel alternative splicing sites; (2) offer a convenient two-level analysis: genome-scale annotation for users with high performance computing environment, and gene-scale annotation for users with personal computers; (3) generate a user-friendly web report and additional BED files for IGV visualization. SplicingTypesAnno is a user-friendly R package for extracting, annotating and analyzing alternative splicing types at exon/intron level for sequence alignment files from RNA-Seq. It is publically available at https://sourceforge.net/projects/splicingtypes/files/ or http://genome.sdau.edu.cn/research/software/SplicingTypesAnno.html. PMID:25720307

  19. The splice is right: Guarantors of fidelity in pre-mRNA splicing

    PubMed Central

    Horowitz, David S.

    2011-01-01

    Two recent papers, one from the Staley laboratory (Koodathingal and colleagues) and the other from the Cheng laboratory (Tseng and colleagues), show that the RNA-dependent ATPase Prp16, which is required for the second step of splicing, acts to reject slowly splicing pre-mRNAs immediately before the first catalytic reaction in pre-mRNA splicing. The results answer long-investigated questions about the actions of Prp16 and provide a wealth of molecular details on the proofreading process in pre-mRNA splicing. The discussion here reviews and integrates the results of the two papers and describes the implications for proofreading in splicing. PMID:21357751

  20. A general definition and nomenclature for alternative splicing events.

    PubMed

    Sammeth, Michael; Foissac, Sylvain; Guigó, Roderic

    2008-01-01

    Understanding the molecular mechanisms responsible for the regulation of the transcriptome present in eukaryotic cells is one of the most challenging tasks in the postgenomic era. In this regard, alternative splicing (AS) is a key phenomenon contributing to the production of different mature transcripts from the same primary RNA sequence. As a plethora of different transcript forms is available in databases, a first step to uncover the biology that drives AS is to identify the different types of reflected splicing variation. In this work, we present a general definition of the AS event along with a notation system that involves the relative positions of the splice sites. This nomenclature univocally and dynamically assigns a specific "AS code" to every possible pattern of splicing variation. On the basis of this definition and the corresponding codes, we have developed a computational tool (AStalavista) that automatically characterizes the complete landscape of AS events in a given transcript annotation of a genome, thus providing a platform to investigate the transcriptome diversity across genes, chromosomes, and species. Our analysis reveals that a substantial part--in human more than a quarter-of the observed splicing variations are ignored in common classification pipelines. We have used AStalavista to investigate and to compare the AS landscape of different reference annotation sets in human and in other metazoan species and found that proportions of AS events change substantially depending on the annotation protocol, species-specific attributes, and coding constraints acting on the transcripts. The AStalavista system therefore provides a general framework to conduct specific studies investigating the occurrence, impact, and regulation of AS. PMID:18688268

  1. Cutting, Splicing, and Kelvin Waves

    NASA Astrophysics Data System (ADS)

    Scheeler, Martin; Kleckner, Dustin; Irvine, William T. M.

    2013-11-01

    Recent experimental advances have allowed us to create, visualize and track vortices of prescribed shape and topology in classical fluids. We study the effect of surgery (cutting and splicing) on the evolution of the geometry and topology of these vortex loops, with a particular focus on the wave-like excitations generated by these operations. We interpret the dynamics of these excitations and the role they play within the broader context of vortex evolution. This work was supported by the National Science Foundation Materials Research and Engineering Centers (MRSEC) Program at the University of Chicago (DMR-0820054) and the Packard Foundation through a Packard fellowship.

  2. Mechanical Splice For Permanent Single Mode And Multimode Fiber Optic Installation

    NASA Astrophysics Data System (ADS)

    Hoffman, A. J.

    1986-01-01

    Over the past three years, GTE Fiber Optic Products has been conducting extensive environmental tests on the Elastomeric Splice to verify its performance in the harsh conditions encountered in the field. These tests include thermal cycling from -40C to +70C, fungus resistance per MIL-STD-810C (Method 508.2), heat/material aging based on activation energies, and blind vs. optimized loss. The results of these tests, which are presented herein, sub-stantiate that the GTE Elastomeric Splice is a proven alternative to fusion and other mechanical splicing systems for both permanent installation as well as emergency repair of multimode and singlemode fiber optic installations.

  3. Splicing factor SRSF1 negatively regulates alternative splicing of MDM2 under damage

    PubMed Central

    Comiskey, Daniel F.; Jacob, Aishwarya G.; Singh, Ravi K.; Tapia-Santos, Aixa S.; Chandler, Dawn S.

    2015-01-01

    Genotoxic stress induces alternative splicing of the oncogene MDM2 generating MDM2-ALT1, an isoform attributed with tumorigenic properties. However, the mechanisms underlying this event remain unclear. Here we explore MDM2 splicing regulation by utilizing a novel minigene that mimics endogenous MDM2 splicing in response to UV and cisplatinum-induced DNA damage. We report that exon 11 is necessary and sufficient for the damage-specific alternative splicing of the MDM2 minigene and that the splicing factor SRSF1 binds exon 11 at evolutionarily conserved sites. Interestingly, mutations disrupting this interaction proved sufficient to abolish the stress-induced alternative splicing of the MDM2 minigene. Furthermore, SRSF1 overexpression promoted exclusion of exon 11, while its siRNA-mediated knockdown prevented the stress-induced alternative splicing of endogenous MDM2. Additionally, we observed elevated SRSF1 levels under stress and in tumors correlating with the expression of MDM2-ALT1. Notably, we demonstrate that MDM2-ALT1 splicing can be blocked by targeting SRSF1 sites on exon 11 using antisense oligonucleotides. These results present conclusive evidence supporting a negative role for SRSF1 in MDM2 alternative splicing. Importantly, we define for the first time, a clear-cut mechanism for the regulation of damage-induced MDM2 splicing and present potential strategies for manipulating MDM2 expression via splicing modulation. PMID:25845590

  4. Functional selection of splicing enhancers that stimulate trans-splicing in vitro.

    PubMed Central

    Boukis, L A; Bruzik, J P

    2001-01-01

    The role of exonic sequences in naturally occurring trans-splicing has not been explored in detail. Here, we have identified trans-splicing enhancers through the use of an iterative selection scheme. Several classes of enhancer sequences were identified that led to dramatic increases in trans-splicing efficiency. Two sequence families were investigated in detail. These include motifs containing the element (G/C)GAC(G/C) and also 5' splice site-like sequences. Distinct elements were tested for their ability to function as splicing enhancers and in competition experiments. In addition, discrete trans-acting factors were identified. This work demonstrates that splicing enhancers are able to effect a large increase in trans-splicing efficiency and that the process of exon definition is able to positively enhance trans-splicing even though the reaction itself is independent of the need for the 5' end of U1 snRNA. Due to the presence of internal introns in messages that are trans-spliced, the natural arrangement of 5' splice sites downstream of trans-splicing acceptors may lead to a general promotion of this unusual reaction. PMID:11421358

  5. Aberrant RNA splicing in cancer; expression changes and driver mutations of splicing factor genes.

    PubMed

    Sveen, A; Kilpinen, S; Ruusulehto, A; Lothe, R A; Skotheim, R I

    2016-05-12

    Alternative splicing is a widespread process contributing to structural transcript variation and proteome diversity. In cancer, the splicing process is commonly disrupted, resulting in both functional and non-functional end-products. Cancer-specific splicing events are known to contribute to disease progression; however, the dysregulated splicing patterns found on a genome-wide scale have until recently been less well-studied. In this review, we provide an overview of aberrant RNA splicing and its regulation in cancer. We then focus on the executors of the splicing process. Based on a comprehensive catalog of splicing factor encoding genes and analyses of available gene expression and somatic mutation data, we identify cancer-associated patterns of dysregulation. Splicing factor genes are shown to be significantly differentially expressed between cancer and corresponding normal samples, and to have reduced inter-individual expression variation in cancer. Furthermore, we identify enrichment of predicted cancer-critical genes among the splicing factors. In addition to previously described oncogenic splicing factor genes, we propose 24 novel cancer-critical splicing factors predicted from somatic mutations. PMID:26300000

  6. Subgroup Specific Alternative Splicing in Medulloblastoma

    PubMed Central

    Kloosterhof, Nanne K; Northcott, Paul A; Yu, Emily PY; Shih, David; Peacock, John; Grajkowska, Wieslawa; van Meter, Timothy; Eberhart, Charles G; Pfister, Stefan; Marra, Marco A; Weiss, William A; Scherer, Stephen W; Rutka, James T; French, Pim J; Taylor, Michael D

    2014-01-01

    Medulloblastoma is comprised of four distinct molecular variants: WNT, SHH, Group 3, and Group 4. We analyzed alternative splicing usage in 14 normal cerebellar samples and 103 medulloblastomas of known subgroup. Medulloblastoma samples have a statistically significant increase in alternative splicing as compared to normal fetal cerebella (2.3-times; P<6.47E-8). Splicing patterns are distinct and specific between molecular subgroups. Unsupervised hierarchical clustering of alternative splicing events accurately assigns medulloblastomas to their correct subgroup. Subgroup-specific splicing and alternative promoter usage was most prevalent in Group 3 (19.4%) and SHH (16.2%) medulloblastomas, while observed less frequently in WNT (3.2%), and Group 4 (9.3%) tumors. Functional annotation of alternatively spliced genes reveals over-representation of genes important for neuronal development. Alternative splicing events in medulloblastoma may be regulated in part by the correlative expression of antisense transcripts, suggesting a possible mechanism affecting subgroup specific alternative splicing. Our results identify additional candidate markers for medulloblastoma subgroup affiliation, further support the existence of distinct subgroups of the disease, and demonstrate an additional level of transcriptional heterogeneity between medulloblastoma subgroups. PMID:22358458

  7. The Evolutionary Fate of Alternatively Spliced Homologous Exons after Gene Duplication

    PubMed Central

    Abascal, Federico; Tress, Michael L.; Valencia, Alfonso

    2015-01-01

    Alternative splicing and gene duplication are the two main processes responsible for expanding protein functional diversity. Although gene duplication can generate new genes and alternative splicing can introduce variation through alternative gene products, the interplay between the two processes is complex and poorly understood. Here, we have carried out a study of the evolution of alternatively spliced exons after gene duplication to better understand the interaction between the two processes. We created a manually curated set of 97 human genes with mutually exclusively spliced homologous exons and analyzed the evolution of these exons across five distantly related vertebrates (lamprey, spotted gar, zebrafish, fugu, and coelacanth). Most of these exons had an ancient origin (more than 400 Ma). We found examples supporting two extreme evolutionary models for the behaviour of homologous axons after gene duplication. We observed 11 events in which gene duplication was accompanied by splice isoform separation, that is, each paralog specifically conserved just one distinct ancestral homologous exon. At other extreme, we identified genes in which the homologous exons were always conserved within paralogs, suggesting that the alternative splicing event cannot easily be separated from the function in these genes. That many homologous exons fall in between these two extremes highlights the diversity of biological systems and suggests that the subtle balance between alternative splicing and gene duplication is adjusted to the specific cellular context of each gene. PMID:25931610

  8. A dynamic alternative splicing program regulates gene expression during terminal erythropoiesis

    PubMed Central

    Pimentel, Harold; Parra, Marilyn; Gee, Sherry; Ghanem, Dana; An, Xiuli; Li, Jie; Mohandas, Narla; Pachter, Lior; Conboy, John G.

    2014-01-01

    Alternative pre-messenger RNA splicing remodels the human transcriptome in a spatiotemporal manner during normal development and differentiation. Here we explored the landscape of transcript diversity in the erythroid lineage by RNA-seq analysis of five highly purified populations of morphologically distinct human erythroblasts, representing the last four cell divisions before enucleation. In this unique differentiation system, we found evidence of an extensive and dynamic alternative splicing program encompassing genes with many diverse functions. Alternative splicing was particularly enriched in genes controlling cell cycle, organelle organization, chromatin function and RNA processing. Many alternative exons exhibited differentiation-associated switches in splicing efficiency, mostly in late-stage polychromatophilic and orthochromatophilic erythroblasts, in concert with extensive cellular remodeling that precedes enucleation. A subset of alternative splicing switches introduces premature translation termination codons into selected transcripts in a differentiation stage-specific manner, supporting the hypothesis that alternative splicing-coupled nonsense-mediated decay contributes to regulation of erythroid-expressed genes as a novel part of the overall differentiation program. We conclude that a highly dynamic alternative splicing program in terminally differentiating erythroblasts plays a major role in regulating gene expression to ensure synthesis of appropriate proteome at each stage as the cells remodel in preparation for production of mature red cells. PMID:24442673

  9. A dynamic alternative splicing program regulates gene expression during terminal erythropoiesis.

    PubMed

    Pimentel, Harold; Parra, Marilyn; Gee, Sherry; Ghanem, Dana; An, Xiuli; Li, Jie; Mohandas, Narla; Pachter, Lior; Conboy, John G

    2014-04-01

    Alternative pre-messenger RNA splicing remodels the human transcriptome in a spatiotemporal manner during normal development and differentiation. Here we explored the landscape of transcript diversity in the erythroid lineage by RNA-seq analysis of five highly purified populations of morphologically distinct human erythroblasts, representing the last four cell divisions before enucleation. In this unique differentiation system, we found evidence of an extensive and dynamic alternative splicing program encompassing genes with many diverse functions. Alternative splicing was particularly enriched in genes controlling cell cycle, organelle organization, chromatin function and RNA processing. Many alternative exons exhibited differentiation-associated switches in splicing efficiency, mostly in late-stage polychromatophilic and orthochromatophilic erythroblasts, in concert with extensive cellular remodeling that precedes enucleation. A subset of alternative splicing switches introduces premature translation termination codons into selected transcripts in a differentiation stage-specific manner, supporting the hypothesis that alternative splicing-coupled nonsense-mediated decay contributes to regulation of erythroid-expressed genes as a novel part of the overall differentiation program. We conclude that a highly dynamic alternative splicing program in terminally differentiating erythroblasts plays a major role in regulating gene expression to ensure synthesis of appropriate proteome at each stage as the cells remodel in preparation for production of mature red cells. PMID:24442673

  10. The Musashi 1 Controls the Splicing of Photoreceptor-Specific Exons in the Vertebrate Retina

    PubMed Central

    Murphy, Daniel; Carstens, Russ

    2016-01-01

    Alternative pre-mRNA splicing expands the coding capacity of eukaryotic genomes, potentially enabling a limited number of genes to govern the development of complex anatomical structures. Alternative splicing is particularly prevalent in the vertebrate nervous system, where it is required for neuronal development and function. Here, we show that photoreceptor cells, a type of sensory neuron, express a characteristic splicing program that affects a broad set of transcripts and is initiated prior to the development of the light sensing outer segments. Surprisingly, photoreceptors lack prototypical neuronal splicing factors and their splicing profile is driven to a significant degree by the Musashi 1 (MSI1) protein. A striking feature of the photoreceptor splicing program are exons that display a "switch-like" pattern of high inclusion levels in photoreceptors and near complete exclusion outside of the retina. Several ubiquitously expressed genes that are involved in the biogenesis and function of primary cilia produce highly photoreceptor specific isoforms through use of such “switch-like” exons. Our results suggest a potential role for alternative splicing in the development of photoreceptors and the conversion of their primary cilia to the light sensing outer segments. PMID:27541351

  11. The Musashi 1 Controls the Splicing of Photoreceptor-Specific Exons in the Vertebrate Retina.

    PubMed

    Murphy, Daniel; Cieply, Benjamin; Carstens, Russ; Ramamurthy, Visvanathan; Stoilov, Peter

    2016-08-01

    Alternative pre-mRNA splicing expands the coding capacity of eukaryotic genomes, potentially enabling a limited number of genes to govern the development of complex anatomical structures. Alternative splicing is particularly prevalent in the vertebrate nervous system, where it is required for neuronal development and function. Here, we show that photoreceptor cells, a type of sensory neuron, express a characteristic splicing program that affects a broad set of transcripts and is initiated prior to the development of the light sensing outer segments. Surprisingly, photoreceptors lack prototypical neuronal splicing factors and their splicing profile is driven to a significant degree by the Musashi 1 (MSI1) protein. A striking feature of the photoreceptor splicing program are exons that display a "switch-like" pattern of high inclusion levels in photoreceptors and near complete exclusion outside of the retina. Several ubiquitously expressed genes that are involved in the biogenesis and function of primary cilia produce highly photoreceptor specific isoforms through use of such "switch-like" exons. Our results suggest a potential role for alternative splicing in the development of photoreceptors and the conversion of their primary cilia to the light sensing outer segments. PMID:27541351

  12. Alternative splicing of the maize Ac transposase transcript in transgenic sugar beet (Beta vulgaris L.)

    PubMed Central

    Lisson, Ralph; Hellert, Jan; Ringleb, Malte; Machens, Fabian; Kraus, Josef

    2010-01-01

    The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT–PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain. PMID:20512402

  13. Alternative splicing of the maize Ac transposase transcript in transgenic sugar beet (Beta vulgaris L.).

    PubMed

    Lisson, Ralph; Hellert, Jan; Ringleb, Malte; Machens, Fabian; Kraus, Josef; Hehl, Reinhard

    2010-09-01

    The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT-PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain. PMID:20512402

  14. The evolutionary fate of alternatively spliced homologous exons after gene duplication.

    PubMed

    Abascal, Federico; Tress, Michael L; Valencia, Alfonso

    2015-06-01

    Alternative splicing and gene duplication are the two main processes responsible for expanding protein functional diversity. Although gene duplication can generate new genes and alternative splicing can introduce variation through alternative gene products, the interplay between the two processes is complex and poorly understood. Here, we have carried out a study of the evolution of alternatively spliced exons after gene duplication to better understand the interaction between the two processes. We created a manually curated set of 97 human genes with mutually exclusively spliced homologous exons and analyzed the evolution of these exons across five distantly related vertebrates (lamprey, spotted gar, zebrafish, fugu, and coelacanth). Most of these exons had an ancient origin (more than 400 Ma). We found examples supporting two extreme evolutionary models for the behaviour of homologous axons after gene duplication. We observed 11 events in which gene duplication was accompanied by splice isoform separation, that is, each paralog specifically conserved just one distinct ancestral homologous exon. At other extreme, we identified genes in which the homologous exons were always conserved within paralogs, suggesting that the alternative splicing event cannot easily be separated from the function in these genes. That many homologous exons fall in between these two extremes highlights the diversity of biological systems and suggests that the subtle balance between alternative splicing and gene duplication is adjusted to the specific cellular context of each gene. PMID:25931610

  15. Phosphoregulation of Ire1 RNase splicing activity

    NASA Astrophysics Data System (ADS)

    Prischi, Filippo; Nowak, Piotr R.; Carrara, Marta; Ali, Maruf M. U.

    2014-04-01

    Ire1 is activated in response to accumulation of misfolded proteins within the endoplasmic reticulum as part of the unfolded protein response (UPR). It is a unique enzyme, possessing both kinase and RNase activity that is required for specific splicing of Xbp1 mRNA leading to UPR activation. How phosphorylation impacts on the Ire1 splicing activity is unclear. In this study, we isolate distinct phosphorylated species of Ire1 and assess their effects on RNase splicing both in vitro and in vivo. We find that phosphorylation within the kinase activation loop significantly increases RNase splicing in vitro. Correspondingly, mutants of Ire1 that cannot be phosphorylated on the activation loop show decreased specific Xbp1 and promiscuous RNase splicing activity relative to wild-type Ire1 in cells. These data couple the kinase phosphorylation reaction to the activation state of the RNase, suggesting that phosphorylation of the activation loop is an important step in Ire1-mediated UPR activation.

  16. Exon skipping restores dystrophin expression, but fails to prevent disease progression in later stage dystrophic dko mice

    PubMed Central

    Wu, B; Cloer, C; Lu, P; Milazi, S; Shaban, M; Shah, SN; Marston-Poe, L; Moulton, HM; Lu, QL

    2014-01-01

    Antisense therapy with both chemistries of phosphorodiamidate morpholino oligomers (PMOs) and 2′-O-methyl phosphorothioate has demonstrated the capability to induce dystrophin expression in Duchenne muscular dystrophy (DMD) patients in phase II–III clinical trials with benefit in muscle functions. However, potential of the therapy for DMD at different stages of the disease progression is not understood. In this study, we examined the effect of peptide-conjugated PMO (PPMO)-mediated exon skipping on disease progression of utrophin-dystrophin-deficient mice (dko) of four age groups (21–29, 30–39, 40–49 and 50+ days), representing diseases from early stage to advanced stage with severe kyphosis. Biweekly intravenous (i.v.) administration of the PPMO restored the dystrophin expression in nearly 100% skeletal muscle fibers in all age groups. This was associated with the restoration of dystrophin-associated proteins including functional glycosylated dystroglycan and neuronal nitric synthase. However, therapeutic outcomes clearly depended on severity of the disease at the time the treatment started. The PPMO treatment alleviated the disease pathology and significantly prolonged the life span of the mice receiving treatment at younger age with mild phenotype. However, restoration of high levels of dystrophin expression failed to prevent disease progression to the mice receiving treatment when disease was already at advanced stage. The results could be critical for design of clinical trials with antisense therapy to DMD. PMID:24942628

  17. Cognitive dysfunction in the dystrophin-deficient mouse model of Duchenne muscular dystrophy: A reappraisal from sensory to executive processes.

    PubMed

    Chaussenot, Rémi; Edeline, Jean-Marc; Le Bec, Benoit; El Massioui, Nicole; Laroche, Serge; Vaillend, Cyrille

    2015-10-01

    Duchenne muscular dystrophy (DMD) is associated with language disabilities and deficits in learning and memory, leading to intellectual disability in a patient subpopulation. Recent studies suggest the presence of broader deficits affecting information processing, short-term memory and executive functions. While the absence of the full-length dystrophin (Dp427) is a common feature in all patients, variable mutation profiles may additionally alter distinct dystrophin-gene products encoded by separate promoters. However, the nature of the cognitive dysfunctions specifically associated with the loss of distinct brain dystrophins is unclear. Here we show that the loss of the full-length brain dystrophin in mdx mice does not modify the perception and sensorimotor gating of auditory inputs, as assessed using auditory brainstem recordings and prepulse inhibition of startle reflex. In contrast, both acquisition and long-term retention of cued and trace fear memories were impaired in mdx mice, suggesting alteration in a functional circuit including the amygdala. Spatial learning in the water maze revealed reduced path efficiency, suggesting qualitative alteration in mdx mice learning strategy. However, spatial working memory performance and cognitive flexibility challenged in various behavioral paradigms in water and radial-arm mazes were unimpaired. The full-length brain dystrophin therefore appears to play a role during acquisition of associative learning as well as in general processes involved in memory consolidation, but no overt involvement in working memory and/or executive functions could be demonstrated in spatial learning tasks. PMID:26190833

  18. Characterization of genetic deletions in Becker muscular dystrophy using monoclonal antibodies against a deletion-prone region of dystrophin

    SciTech Connect

    Thanh, L.T.; Man, Nguyen Thi; Morris, G.E.

    1995-08-28

    We have produced a new panel of 20 monoclonal antibodies (mAbs) against a region of the dystrophin protein corresponding to a deletion-prone region of the Duchenne muscular dystrophy gene (exons 45-50). We show that immunohistochemistry or Western blotting with these {open_quotes}exon-specific{close_quotes} mAbs can provide a valuable addition to Southern blotting or PCR methods for the accurate identification of genetic deletions in Becker muscular dystrophy patients. The antibodies were mapped to the following exons: exon 45 (2 mAbs), exon 46 (6), exon 47 (1), exons 47/48 (4), exons 48-50 (6), and exon 50 (1). PCR amplification of single exons or groups of exons was used both to produce specific dystrophin immunogens and to map the mAbs obtained. PCR-mediated mutagenesis was also used to identify regions of dystrophin important for mAb binding. Because the mAbs can be used to characterize the dystrophin produced by individual muscle fibres, they will also be useful for studying {open_quotes}revertant{close_quotes} fibres in Duchenne muscle and for monitoring the results of myoblast therapy trials in MD patients with deletions in this region of the dystrophin gene. 27 refs., 7 figs., 3 tabs.

  19. Becker Muscular Dystrophy (BMD) caused by duplication of exons 3-6 of the dystrophin gene presenting as dilated cardiomyopathy

    SciTech Connect

    Tsai, A.C.; Allingham-Hawkins, D.J.; Becker, L.

    1994-09-01

    X-linked dilated cardiomyopathy (XLCM) is a progressive myocardial disease presenting with congestive heart failure in teenage males without clinical signs of skeletal myopathy. Tight linkage of XLCM to the DMD locus has been demonstrated; it has been suggested that, at least in some families, XLCM is a {open_quotes}dystrophinopathy.{close_quotes} We report a 14-year-old boy who presented with acute heart failure due to dilated cardiomyopathy. He had no history of muscle weakness, but physical examination revealed pseudohypertrophy of the calf muscles. He subsequently received a heart transplantation. Family history was negative. Serum CK level at the time of diagnosis was 10,416. Myocardial biopsy showed no evidence of carditis. Dystrophin staining of cardiac and skeletal muscle with anti-sera to COOH and NH{sub 2}termini showed a patchy distribution of positivity suggestive of Becker muscular dystrophy. Analysis of 18 of the 79 dystrophin exons detected a duplication that included exons 3-6. The proband`s mother has an elevated serum CK and was confirmed to be a carrier of the same duplication. A mutation in the muscle promotor region of the dystrophin gene has been implicated in the etiology of SLCM. However, Towbin et al. (1991) argued that other 5{prime} mutations in the dystrophin gene could cause selective cardiomyopathy. The findings in our patient support the latter hypothesis. This suggests that there are multiple regions in the dystrophin gene which, when disrupted, can cause isolated dilated cardiomyopathy.

  20. Security controls in the Stockpoint Logistics Integrated Communications Environment (SPLICE)

    NASA Astrophysics Data System (ADS)

    Arseneault, D. S.

    1985-03-01

    This thesis examines security controls specified and implemented in the Stock Point Logistics Integrated Communications Environment (SPLICE) project. Controls provided by the Defense Data Network and the Tandem operating system are reviewed. Alternatives from current literature in areas of authentication, encryption, and dial-port protection are reviewed for the purpose of suggesting enhancements. Issues discussed apply to most interactive/decentralized systems in operation today and include administrative as well as technical recommendations.

  1. Splicing regulation: From a parts list of regulatory elements to an integrated splicing code

    PubMed Central

    Wang, Zefeng; Burge, Christopher B.

    2008-01-01

    Alternative splicing of pre-mRNAs is a major contributor to both proteomic diversity and control of gene expression levels. Splicing is tightly regulated in different tissues and developmental stages, and its disruption can lead to a wide range of human diseases. An important long-term goal in the splicing field is to determine a set of rules or “code” for splicing that will enable prediction of the splicing pattern of any primary transcript from its sequence. Outside of the core splice site motifs, the bulk of the information required for splicing is thought to be contained in exonic and intronic cis-regulatory elements that function by recruitment of sequence-specific RNA-binding protein factors that either activate or repress the use of adjacent splice sites. Here, we summarize the current state of knowledge of splicing cis-regulatory elements and their context-dependent effects on splicing, emphasizing recent global/genome-wide studies and open questions. PMID:18369186

  2. Role of the 3′ Splice Site in U12-Dependent Intron Splicing

    PubMed Central

    Dietrich, Rosemary C.; Peris, Marian J.; Seyboldt, Andrew S.; Padgett, Richard A.

    2001-01-01

    U12-dependent introns containing alterations of the 3′ splice site AC dinucleotide or alterations in the spacing between the branch site and the 3′ splice site were examined for their effects on splice site selection in vivo and in vitro. Using an intron with a 5′ splice site AU dinucleotide, any nucleotide could serve as the 3′-terminal nucleotide, although a C residue was most active, while a U residue was least active. The penultimate A residue, by contrast, was essential for 3′ splice site function. A branch site-to-3′ splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3′ splice sites. A strong preference for a spacing of about 12 nucleotides was observed. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3′ splice site but that formation of the prespliceosomal complex A requires an active 3′ splice site. Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3′ splice site. PMID:11238930

  3. Automated Eukaryotic Gene Structure Annotation Using EVidenceModeler and the Program to Assemble Spliced Alignments

    SciTech Connect

    Haas, B J; Salzberg, S L; Zhu, W; Pertea, M; Allen, J E; Orvis, J; White, O; Buell, C R; Wortman, J R

    2007-12-10

    EVidenceModeler (EVM) is presented as an automated eukaryotic gene structure annotation tool that reports eukaryotic gene structures as a weighted consensus of all available evidence. EVM, when combined with the Program to Assemble Spliced Alignments (PASA), yields a comprehensive, configurable annotation system that predicts protein-coding genes and alternatively spliced isoforms. Our experiments on both rice and human genome sequences demonstrate that EVM produces automated gene structure annotation approaching the quality of manual curation.

  4. Dystrophin expression following the transplantation of normal muscle precursor cells protects mdx muscle from contraction-induced damage.

    PubMed

    Rousseau, Joel; Dumont, Nicolas; Lebel, Carl; Quenneville, Simon P; Côté, Claude H; Frenette, Jérome; Tremblay, Jacques P

    2010-01-01

    Duchenne muscular dystrophy (DMD) is the most frequent muscular dystrophy. Currently, there is no cure for the disease. The transplantation of muscle precursor cells (MPCs) is one of the possible treatments, because it can restore the expression of dystrophin in DMD muscles. In this study, we investigated the effects of myoblasts injected with cardiotoxin on the contractile properties and resistance to eccentric contractions of transplanted and nontransplanted muscles. We used the extensor digitorum longus (EDL) as a model for our study. We conclude that the sole presence of dystrophin in a high percentage of muscle fibers is not sufficient by itself to increase the absolute or the specific force in the EDL of transplanted mdx muscle. This lack of strength increase may be due to the extensive damage that was produced by the cardiotoxin, which was coinjected with the myoblasts. However, the dystrophin presence is sufficient to protect muscle from eccentric damage as indicated by the force drop results. PMID:20650035

  5. Dystrophin conferral using human endothelium expressing HLA-E in the non-immunosuppressive murine model of Duchenne muscular dystrophy.

    PubMed

    Cui, Chang-Hao; Miyoshi, Shunichiro; Tsuji, Hiroko; Makino, Hatsune; Kanzaki, Seiichi; Kami, Daisuke; Terai, Masanori; Suzuki, Harumi; Umezawa, Akihiro

    2011-01-15

    Human leukocyte antigen (HLA)-E is a non-classical major histocompatibility complex class I (Ib) molecule, which plays an important role in immunosuppression. In this study, we investigated the immunomodulating effect of HLA-E in a xenogeneic system, using human placental artery-derived endothelial (hPAE) cells expressing HLA-E in a mouse model. In vitro cell lysis analysis by primed lymphocytes in combination with siRNA transfection showed that HLA-E is necessary for inhibition of the immune response. Similarly, in vivo cell implantation analysis with siRNA-mediated down-regulation of HLA-E demonstrates that HLA-E is involved in immunosuppression. As hPAE cells efficiently transdifferentiate into myoblasts/myocytes in vitro, we transplanted the cells into mdx mice, a model of Duchenne muscular dystrophy. hPAE cells conferred dystrophin to myocytes of the 'immunocompetent' mdx mice with extremely high efficiency. These findings suggest that HLA-E-expressing cells with a myogenic potential represent a promising source for cell-based therapy of patients with muscular dystrophy. PMID:20947660

  6. Mini- and full-length dystrophin gene transfer induces the recovery of nitric oxide synthase at the sarcolemma of mdx4cv skeletal muscle fibers.

    PubMed

    Decrouy, A; Renaud, J M; Lunde, J A; Dickson, G; Jasmin, B J

    1998-01-01

    In normal skeletal muscle fibers, dystrophin accumulates at the cytoplasmic face of the sarcolemma where it associates with dystrophin-associated proteins (DAPs). Several studies have recently shown that the neuronal isoform of nitric oxide synthase (nNOS) is also located at the sarcolemma, and that this membrane localization is mediated through interactions of nNOS with one of the DAPs, namely alpha 1-syntrophin. Since the lack of dystrophin in muscle fibers from Duchenne muscular dystrophy patients and mdx mice is accompanied by an absence of sarcolemmal nNOS, we examined in the present study, whether dystrophin gene replacement would lead to the restoration of nNOS at its appropriate subcellular location. To this end, tibialis anterior muscles from mdx4cv mice were directly injected with plasmid DNA encoding either full-length (pRSV-dys) or mini-(pRSV-dyB; lacking exons 17-48) dystrophin. For these experiments, we chose to study 10-week-old mdx4cv mice since at this developmental stage, muscles from these mice have already undergone several cycles of degeneration-regeneration. Immunofluorescence experiments performed on serial cross-sections revealed that approximately 50% of the dystrophin-positive fibers also exhibited significant levels of nNOS at their sarcolemma 2 weeks following gene transfer with pRSV-dys. Similar results were obtained with pRSV-dyB indicating that exons 17-48 of the dystrophin gene are not essential for the correct localization of nNOS in skeletal muscle fibers. Taken together with the recent demonstration that dystrophin gene transfer leads to significant physiological benefits our results suggest that dystrophin gene therapy using full-length or truncated dystrophin, also induces a rapid recovery of biochemical functions. PMID:9536265

  7. A Sensitive, Reproducible and Objective Immunofluorescence Analysis Method of Dystrophin in Individual Fibers in Samples from Patients with Duchenne Muscular Dystrophy

    PubMed Central

    Beekman, Chantal; Sipkens, Jessica A.; Testerink, Janwillem; Giannakopoulos, Stavros; Kreuger, Dyonne; van Deutekom, Judith C.; Campion, Giles V.; de Kimpe, Sjef J.; Lourbakos, Afrodite

    2014-01-01

    Duchenne muscular dystrophy (DMD) is characterized by the absence or reduced levels of dystrophin expression on the inner surface of the sarcolemmal membrane of muscle fibers. Clinical development of therapeutic approaches aiming to increase dystrophin levels requires sensitive and reproducible measurement of differences in dystrophin expression in muscle biopsies of treated patients with DMD. This, however, poses a technical challenge due to intra- and inter-donor variance in the occurrence of revertant fibers and low trace dystrophin expression throughout the biopsies. We have developed an immunofluorescence and semi-automated image analysis method that measures the sarcolemmal dystrophin intensity per individual fiber for the entire fiber population in a muscle biopsy. Cross-sections of muscle co-stained for dystrophin and spectrin have been imaged by confocal microscopy, and image analysis was performed using Definiens software. Dystrophin intensity has been measured in the sarcolemmal mask of spectrin for each individual muscle fiber and multiple membrane intensity parameters (mean, maximum, quantiles per fiber) were calculated. A histogram can depict the distribution of dystrophin intensities for the fiber population in the biopsy. This method was tested by measuring dystrophin in DMD, Becker muscular dystrophy, and healthy muscle samples. Analysis of duplicate or quadruplicate sections of DMD biopsies on the same or multiple days, by different operators, or using different antibodies, was shown to be objective and reproducible (inter-assay precision, CV 2–17% and intra-assay precision, CV 2–10%). Moreover, the method was sufficiently sensitive to detect consistently small differences in dystrophin between two biopsies from a patient with DMD before and after treatment with an investigational compound. PMID:25244123

  8. EASI--enrichment of alternatively spliced isoforms.

    PubMed

    Venables, Julian P; Burn, John

    2006-01-01

    Alternative splicing produces more than one protein from the majority of genes and the rarer forms can have dominant functions. Instability of alternative transcripts can also hinder the study of regulation of gene expression by alternative splicing. To investigate the true extent of alternative splicing we have developed a simple method of enriching alternatively spliced isoforms (EASI) from PCRs using beads charged with Thermus aquaticus single-stranded DNA-binding protein (T.Aq ssb). This directly purifies the single-stranded regions of heteroduplexes between alternative splices formed in the PCR, enabling direct sequencing of all the rare alternative splice forms of any gene. As a proof of principle the alternative transcripts of three tumour suppressor genes, TP53, MLH1 and MSH2, were isolated from testis cDNA. These contain missing exons, cryptic splice sites or include completely novel exons. EASI beads are stable for months in the fridge and can be easily combined with standard protocols to speed the cloning of novel transcripts. PMID:16951290

  9. 30 CFR 57.12088 - Splicing trailing cables.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Splicing trailing cables. 57.12088 Section 57... Underground Only § 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its equivalent, shall be made in a trailing cable within 25 feet of the machine unless the machine is equipped with...

  10. 30 CFR 57.12088 - Splicing trailing cables.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Splicing trailing cables. 57.12088 Section 57... Underground Only § 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its equivalent, shall be made in a trailing cable within 25 feet of the machine unless the machine is equipped with...

  11. 30 CFR 57.12088 - Splicing trailing cables.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Splicing trailing cables. 57.12088 Section 57... Underground Only § 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its equivalent, shall be made in a trailing cable within 25 feet of the machine unless the machine is equipped with...

  12. 30 CFR 57.12088 - Splicing trailing cables.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Splicing trailing cables. 57.12088 Section 57... Underground Only § 57.12088 Splicing trailing cables. No splice, except a vulcanized splice or its equivalent, shall be made in a trailing cable within 25 feet of the machine unless the machine is equipped with...

  13. Screening of point mutations by multiple SSCP analysis in the dystrophin gene

    SciTech Connect

    Lasa, A.; Baiget, M.; Gallano, P.

    1994-09-01

    Duchenne muscular dystrophy (DMD) is a lethal, X-linked neuromuscular disorder. The population frequency of DMD is one in approximately 3500 boys, of which one third is thought to be a new mutant. The DMD gene is the largest known to date, spanning over 2,3 Mb in band Xp21.2; 79 exons are transcribed into a 14 Kb mRNA coding for a protein of 427 kD which has been named dystrophin. It has been shown that about 65% of affected boys have a gene deletion with a wide variation in localization and size. The remaining affected individuals who have no detectable deletions or duplications would probably carry more subtle mutations that are difficult to detect. These mutations occur in several different exons and seem to be unique to single patients. Their identification represents a formidable goal because of the large size and complexity of the dystrophin gene. SSCP is a very efficient method for the detection of point mutations if the parameters that affect the separation of the strands are optimized for a particular DNA fragment. The multiple SSCP allows the simultaneous study of several exons, and implies the use of different conditions because no single set of conditions will be optimal for all fragments. Seventy-eight DMD patients with no deletion or duplication in the dystrophin gene were selected for the multiple SSCP analysis. Genomic DNA from these patients was amplified using the primers described for the diagnosis procedure (muscle promoter and exons 3, 8, 12, 16, 17, 19, 32, 45, 48 and 51). We have observed different mobility shifts in bands corresponding to exons 8, 12, 43 and 51. In exons 17 and 45, altered electrophoretic patterns were found in different samples identifying polymorphisms already described.

  14. Aberrant splicing and drug resistance in AML.

    PubMed

    de Necochea-Campion, Rosalia; Shouse, Geoffrey P; Zhou, Qi; Mirshahidi, Saied; Chen, Chien-Shing

    2016-01-01

    The advent of next-generation sequencing technologies has unveiled a new window into the heterogeneity of acute myeloid leukemia (AML). In particular, recurrent mutations in spliceosome machinery and genome-wide aberrant splicing events have been recognized as a prominent component of this disease. This review will focus on how these factors influence drug resistance through altered splicing of tumor suppressor and oncogenes and dysregulation of the apoptotic signaling network. A better understanding of these factors in disease progression is necessary to design appropriate therapeutic strategies recognizing specific alternatively spliced or mutated oncogenic targets. PMID:27613060

  15. Control of neuronal synapse specification by a highly dedicated alternative splicing program.

    PubMed

    Traunmüller, Lisa; Gomez, Andrea M; Nguyen, Thi-Minh; Scheiffele, Peter

    2016-05-20

    Alternative RNA splicing represents a central mechanism for expanding the coding power of genomes. Individual RNA-binding proteins can control alternative splicing choices in hundreds of RNA transcripts, thereby tuning amounts and functions of large numbers of cellular proteins. We found that the RNA-binding protein SLM2 is essential for functional specification of glutamatergic synapses in the mouse hippocampus. Genome-wide mapping revealed a markedly selective SLM2-dependent splicing program primarily consisting of only a few target messenger RNAs that encode synaptic proteins. Genetic correction of a single SLM2-dependent target exon in the synaptic recognition molecule neurexin-1 was sufficient to rescue synaptic plasticity and behavioral defects in Slm2 knockout mice. These findings uncover a highly selective alternative splicing program that specifies synaptic properties in the central nervous system. PMID:27174676

  16. Meayamycin Inhibits pre-mRNA Splicing and Exhibits Picomolar Activity Against Multidrug Resistant Cells

    PubMed Central

    Albert, Brian J.; McPherson, Peter A.; O'Brien, Kristine; Czaicki, Nancy L.; DeStefino, Vincent; Osman, Sami; Li, Miaosheng; Day, Billy W.; Grabowski, Paula J.; Moore, Melissa J.; Vogt, Andreas; Koide, Kazunori

    2009-01-01

    FR901464 is a potent antitumor natural product that binds to the SF3b complex and inhibits pre-mRNA splicing. Its analogue, meayamycin, is two orders of magnitude more potent as an antiproliferative agent against human breast cancer MCF-7 cells. Here, we report the picomolar antiproliferative activity of meayamycin against various cancer cell lines and multidrug resistant cells. Time-dependence studies implied that meayamycin may form a covalent bond with its target protein(s). Meayamycin inhibited pre-mRNA splicing in HEK-293 cells but not alternative splicing in a neuronal system. Meayamycin exhibited specificity toward human lung cancer cells compared to non-tumorigenic human lung fibroblasts and retained picomolar growth inhibitory activity against multi-drug resistant cells. These data suggest that meayamycin is a useful chemical probe to study pre-mRNA splicing in live cells and is a promising lead as an anticancer agent. PMID:19671752

  17. Regulation of Alternative Splicing in Vivo by Overexpression of Antagonistic Splicing Factors

    NASA Astrophysics Data System (ADS)

    Caceres, Javier F.; Stamm, Stefan; Helfman, David M.; Krainer, Adrian R.

    1994-09-01

    The opposing effects of SF2/ASF and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 influence alternative splicing in vitro. SF2/ASF or hnRNP A1 complementary DNAs were transiently overexpressed in HeLa cells, and the effect on alternative splicing of several cotransfected reporter genes was measured. Increased expression of SF2/ASF activated proximal 5' splice sites, promoted inclusion of a neuron-specific exon, and prevented abnormal exon skipping. Increased expression of hnRNP A1 activated distal 5' splice sites. Therefore, variations in the intracellular levels of antagonistic splicing factors influence different modes of alternative splicing in vivo and may be a natural mechanism for tissue-specific or developmental regulation of gene expression.

  18. RNA-Binding Proteins: Splicing Factors and Disease

    PubMed Central

    Fredericks, Alger M.; Cygan, Kamil J.; Brown, Brian A.; Fairbrother, William G.

    2015-01-01

    Pre-mRNA splicing is mediated by interactions of the Core Spliceosome and an array of accessory RNA binding proteins with cis-sequence elements. Splicing is a major regulatory component in higher eukaryotes. Disruptions in splicing are a major contributor to human disease. One in three hereditary disease alleles are believed to cause aberrant splicing. Hereditary disease alleles can alter splicing by disrupting a splicing element, creating a toxic RNA, or affecting splicing factors. One of the challenges of medical genetics is identifying causal variants from the thousands of possibilities discovered in a clinical sequencing experiment. Here we review the basic biochemistry of splicing, the mechanisms of splicing mutations, the methods for identifying splicing mutants, and the potential of therapeutic interventions. PMID:25985083

  19. A 32-nucleotide exon-splicing enhancer regulates usage of competing 5' splice sites in a differential internal exon.

    PubMed Central

    Humphrey, M B; Bryan, J; Cooper, T A; Berget, S M

    1995-01-01

    Large alternatively spliced internal exons are uncommon in vertebrate genes, and the mechanisms governing their usage are unknown. In this report, we examined alternative splicing of a 1-kb internal exon from the human caldesmon gene containing two regulated 5' splice sites that are 687 nucleotides apart. In cell lines normally splicing caldesmon RNA via utilization of the exon-internal 5' splice site, inclusion of the differential exon required a long purine-rich sequence located between the two competing 5' splice sites. This element consisted of four identical 32-nucleotide purine-rich repeats that resemble exon-splicing enhancers (ESE) identified in other genes. One 32-nucleotide repeat supported exon inclusion, repressed usage of the terminal 5' splice site, and functioned in a heterologous exon dependent on exon enhancers for inclusion, indicating that the caldesmon purine-rich sequence can be classified as an ESE. The ESE was required for utilization of the internal 5' splice site only in the presence of the competing 5' splice site and had no effect when placed downstream of the terminal 5' splice site. In the absence of the internal 5' splice site, the ESE activated a normally silent cryptic 5' splice site near the natural internal 5' splice site, indicating that the ESE stimulates upstream 5' splice site selection. We propose that the caldesmon ESE functions to regulate competition between two 5' splice sites within a differential internal exon. PMID:7623794

  20. Competition between pre-mRNAs for the splicing machinery drives global regulation of splicing.

    PubMed

    Munding, Elizabeth M; Shiue, Lily; Katzman, Sol; Donohue, John Paul; Ares, Manuel

    2013-08-01

    During meiosis in yeast, global splicing efficiency increases and then decreases. Here we provide evidence that splicing improves due to reduced competition for the splicing machinery. The timing of this regulation corresponds to repression and reactivation of ribosomal protein genes (RPGs) during meiosis. In vegetative cells, RPG repression by rapamycin treatment also increases splicing efficiency. Downregulation of the RPG-dedicated transcription factor gene IFH1 genetically suppresses two spliceosome mutations, prp11-1 and prp4-1, and globally restores splicing efficiency in prp4-1 cells. We conclude that the splicing apparatus is limiting and that pre-messenger RNAs compete. Splicing efficiency of a pre-mRNA therefore depends not just on its own concentration and affinity for limiting splicing factor(s), but also on those of competing pre-mRNAs. Competition between RNAs for limiting processing factors appears to be a general condition in eukaryotes for a variety of posttranscriptional control mechanisms including microRNA (miRNA) repression, polyadenylation, and splicing. PMID:23891561

  1. A Gene Gun-mediated Nonviral RNA trans-splicing Strategy for Col7a1 Repair.

    PubMed

    Peking, Patricia; Koller, Ulrich; Hainzl, Stefan; Kitzmueller, Sophie; Kocher, Thomas; Mayr, Elisabeth; Nyström, Alexander; Lener, Thomas; Reichelt, Julia; Bauer, Johann W; Murauer, Eva M

    2016-01-01

    RNA trans-splicing represents an auspicious option for the correction of genetic mutations at RNA level. Mutations within COL7A1 causing strong reduction or absence of type VII collagen are associated with the severe skin blistering disease dystrophic epidermolysis bullosa. The human COL7A1 mRNA constitutes a suitable target for this RNA therapy approach, as only a portion of the almost 9 kb transcript has to be delivered into the target cells. Here, we have proven the feasibility of 5' trans-splicing into the Col7a1 mRNA in vitro and in vivo. We designed a 5' RNA trans-splicing molecule, capable of replacing Col7a1 exons 1-15 and verified it in a fluorescence-based trans-splicing model system. Specific and efficient Col7a1 trans-splicing was confirmed in murine keratinocytes. To analyze trans-splicing in vivo, we used gene gun delivery of a minicircle expressing a FLAG-tagged 5' RNA trans-splicing molecule into the skin of wild-type mice. Histological and immunofluorescence analysis of bombarded skin sections revealed vector delivery and expression within dermis and epidermis. Furthermore, we have detected trans-spliced type VII collagen protein using FLAG-tag antibodies. In conclusion, we describe a novel in vivo nonviral RNA therapy approach to restore type VII collagen expression for causative treatment of dystrophic epidermolysis bullosa. PMID:26928235

  2. Group II Intron Self-Splicing.

    PubMed

    Pyle, Anna Marie

    2016-07-01

    Group II introns are large, autocatalytic ribozymes that catalyze RNA splicing and retrotransposition. Splicing by group II introns plays a major role in the metabolism of plants, fungi, and yeast and contributes to genetic variation in many bacteria. Group II introns have played a major role in genome evolution, as they are likely progenitors of spliceosomal introns, retroelements, and other machinery that controls genetic variation and stability. The structure and catalytic mechanism of group II introns have recently been elucidated through a combination of genetics, chemical biology, solution biochemistry, and crystallography. These studies reveal a dynamic machine that cycles progressively through multiple conformations as it stimulates the various stages of splicing. A central active site, containing a reactive metal ion cluster, catalyzes both steps of self-splicing. These studies provide insights into RNA structure, folding, and catalysis, as they raise new questions about the behavior of RNA machines. PMID:27391926

  3. Lessons from non-canonical splicing.

    PubMed

    Sibley, Christopher R; Blazquez, Lorea; Ule, Jernej

    2016-07-01

    Recent improvements in experimental and computational techniques that are used to study the transcriptome have enabled an unprecedented view of RNA processing, revealing many previously unknown non-canonical splicing events. This includes cryptic events located far from the currently annotated exons and unconventional splicing mechanisms that have important roles in regulating gene expression. These non-canonical splicing events are a major source of newly emerging transcripts during evolution, especially when they involve sequences derived from transposable elements. They are therefore under precise regulation and quality control, which minimizes their potential to disrupt gene expression. We explain how non-canonical splicing can lead to aberrant transcripts that cause many diseases, and also how it can be exploited for new therapeutic strategies. PMID:27240813

  4. Cell penetrating peptide delivery of splice directing oligonucleotides as a treatment for Duchenne muscular dystrophy.

    PubMed

    Betts, Corinne A; Wood, Matthew J A

    2013-01-01

    Duchenne muscular dystrophy is a severe, X-linked muscle wasting disorder caused by the absence of an integral structural protein called dystrophin. This is caused by mutations or deletions in the dystrophin gene which disrupt the reading frame, thereby halting the production of a functional protein. A number of potential therapies have been investigated for the treatment of this disease including utrophin upregulation, 'stop-codon read through' aminoglycosides and adeno-associated virus gene replacement as well as stem cell therapy. However, the most promising treatment to date is the use of antisense oligonucleotides which cause exon skipping by binding to a specific mRNA sequence, skipping the desired exon, thereby restoring the reading frame and producing a truncated yet functional protein. The results from recent 2'OMePS and morpholino clinical trials have renewed hope for Duchenne patients; however in vivo studies in a mouse model, mdx, have revealed low systemic distribution and poor delivery of oligonucleotides to affected tissues such as the brain and heart. However a variety of cell penetrating peptides directly conjugated to antisense oligonucleotides have been shown to enhance delivery in Duchenne model systems with improved systemic distribution and greater efficacy compared to 'naked' antisense oligonucleotides. These cell penetrating peptides, combined with an optimised dose and dosing regimen, as well as thorough toxicity profile have the potential to be developed into a promising treatment which may be progressed to clinical trial. PMID:23140454

  5. Evolutionary connections between coding and splicing regulatory regions in the fibronectin EDA exon.

    PubMed

    Zago, Paola; Buratti, Emanuele; Stuani, Cristiana; Baralle, Francisco E

    2011-08-01

    Research on exonic coding sequences has demonstrated that many substitutions at the amino acid level may also reflect profound changes at the level of splicing regulatory regions. These results have revealed that, for many alternatively spliced exons, there is considerable pressure to strike a balance between two different and sometimes conflicting forces: the drive to improve the quality and production efficiency of proteins and the maintenance of proper exon recognition by the splicing machinery. Up to now, the systems used to investigate these connections have mostly focused on short alternatively spliced exons that contain a high density of splicing regulatory elements. Although this is obviously a desirable feature in order to maximize the chances of spotting connections, it also complicates the process of drawing straightforward evolutionary pathways between different species (because of the numerous alternative pathways through which the same end point can be achieved). The alternatively spliced fibronectin extra domain A exon (also referred to as EDI or EIIIA) does not have these limitations, as its inclusion is already known to depend on a single exonic splicing enhancer element within its sequence. In this study, we have compared the rat and human fibronectin EDA exons with regard to RNA structure, exonic splicing enhancer strengths, and SR protein occupancy. The results gained from these analyses have then been used to perform an accurate evaluation of EDA sequences observed in a wide range of animal species. This comparison strongly suggests the existence of an evolutionary connection between changes at the nucleotide levels and the need to maintain efficient EDA recognition in different species. PMID:21663748

  6. Complex genomic rearrangements in the dystrophin gene due to replication-based mechanisms

    PubMed Central

    Baskin, Berivan; Stavropoulos, Dimitri J; Rebeiro, Paige A; Orr, Jennifer; Li, Martin; Steele, Leslie; Marshall, Christian R; Lemire, Edmond G; Boycott, Kym M; Gibson, William; Ray, Peter N

    2014-01-01

    Genomic rearrangements such as intragenic deletions and duplications are the most prevalent type of mutations in the dystrophin gene resulting in Duchenne and Becker muscular dystrophy (D/BMD). These copy number variations (CNVs) are nonrecurrent and can result from either nonhomologous end joining (NHEJ) or microhomology-mediated replication-dependent recombination (MMRDR). We characterized five DMD patients with complex genomic rearrangements using a combination of MLPA/mRNA transcript analysis/custom array comparative hybridization arrays (CGH) and breakpoint sequence analysis to investigate the mechanisms for these rearrangements. Two patients had complex rearrangements that involved microhomologies at breakpoints. One patient had a noncontiguous insertion of 89.7 kb chromosome 4 into intron 43 of DMD involving three breakpoints with 2–5 bp microhomology at the junctions. A second patient had an inversion of exon 44 flanked by intronic deletions with two breakpoint junctions each showing 2 bp microhomology. The third patient was a female with an inherited deletion of exon 47 in DMD on the maternal allele and a de novo noncontiguous duplication of exons 45–49 in DMD and MID1 on the paternal allele. The other two patients harbored complex noncontiguous duplications within the dystrophin gene. We propose a replication-based mechanisms for all five complex DMD rearrangements. This study identifies additional underlying mechanisms in DMD, and provides insight into the molecular bases of these genomic rearrangements. PMID:25614876

  7. Characterization of Dystrophin Deficient Rats: A New Model for Duchenne Muscular Dystrophy

    PubMed Central

    Tesson, Laurent; Remy, Séverine; Thepenier, Virginie; François, Virginie; Le Guiner, Caroline; Goubin, Helicia; Dutilleul, Maéva; Guigand, Lydie; Toumaniantz, Gilles; De Cian, Anne; Boix, Charlotte; Renaud, Jean-Baptiste; Cherel, Yan; Giovannangeli, Carine; Concordet, Jean-Paul; Anegon, Ignacio; Huchet, Corinne

    2014-01-01

    A few animal models of Duchenne muscular dystrophy (DMD) are available, large ones such as pigs or dogs being expensive and difficult to handle. Mdx (X-linked muscular dystrophy) mice only partially mimic the human disease, with limited chronic muscular lesions and muscle weakness. Their small size also imposes limitations on analyses. A rat model could represent a useful alternative since rats are small animals but 10 times bigger than mice and could better reflect the lesions and functional abnormalities observed in DMD patients. Two lines of Dmd mutated-rats (Dmdmdx) were generated using TALENs targeting exon 23. Muscles of animals of both lines showed undetectable levels of dystrophin by western blot and less than 5% of dystrophin positive fibers by immunohistochemistry. At 3 months, limb and diaphragm muscles from Dmdmdx rats displayed severe necrosis and regeneration. At 7 months, these muscles also showed severe fibrosis and some adipose tissue infiltration. Dmdmdx rats showed significant reduction in muscle strength and a decrease in spontaneous motor activity. Furthermore, heart morphology was indicative of dilated cardiomyopathy associated histologically with necrotic and fibrotic changes. Echocardiography showed significant concentric remodeling and alteration of diastolic function. In conclusion, Dmdmdx rats represent a new faithful small animal model of DMD. PMID:25310701

  8. Nonmechanical Roles of Dystrophin and Associated Proteins in Exercise, Neuromuscular Junctions, and Brains

    PubMed Central

    Nichols, Bailey; Takeda, Shin’ichi; Yokota, Toshifumi

    2015-01-01

    Dystrophin-glycoprotein complex (DGC) is an important structural unit in skeletal muscle that connects the cytoskeleton (f-actin) of a muscle fiber to the extracellular matrix (ECM). Several muscular dystrophies, such as Duchenne muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophies (dystroglycanopathies), and limb-girdle muscular dystrophies (sarcoglycanopathies), are caused by mutations in the different DGC components. Although many early studies indicated DGC plays a crucial mechanical role in maintaining the structural integrity of skeletal muscle, recent studies identified novel roles of DGC. Beyond a mechanical role, these DGC members play important signaling roles and act as a scaffold for various signaling pathways. For example, neuronal nitric oxide synthase (nNOS), which is localized at the muscle membrane by DGC members (dystrophin and syntrophins), plays an important role in the regulation of the blood flow during exercise. DGC also plays important roles at the neuromuscular junction (NMJ) and in the brain. In this review, we will focus on recently identified roles of DGC particularly in exercise and the brain. PMID:26230713

  9. Losartan decreases cardiac muscle fibrosis and improves cardiac function in dystrophin-deficient mdx mice.

    PubMed

    Spurney, Christopher F; Sali, Arpana; Guerron, Alfredo D; Iantorno, Micaela; Yu, Qing; Gordish-Dressman, Heather; Rayavarapu, Sree; van der Meulen, Jack; Hoffman, Eric P; Nagaraju, Kanneboyina

    2011-03-01

    Recent studies showed that chronic administration of losartan, an angiotensin II type I receptor antagonist, improved skeletal muscle function in dystrophin-deficient mdx mice. In this study, C57BL/10ScSn-Dmd(mdx)/J female mice were either untreated or treated with losartan (n = 15) in the drinking water at a dose of 600 mg/L over a 6-month period. Cardiac function was assessed via in vivo high frequency echocardiography and skeletal muscle function was assessed using grip strength testing, Digiscan monitoring, Rotarod timing, and in vitro force testing. Fibrosis was assessed using picrosirius red staining and Image J analysis. Gene expression was evaluated using real-time polymerized chain reaction (RT-PCR). Percentage shortening fraction was significantly decreased in untreated (26.9% ± 3.5%) mice compared to losartan-treated (32.2% ± 4.2%; P < .01) mice. Systolic blood pressure was significantly reduced in losartan-treated mice (56 ± 6 vs 69 ± 7 mm Hg; P < .0005). Percentage cardiac fibrosis was significantly reduced in losartan-treated hearts (P < .05) along with diaphragm (P < .01), extensor digitorum longus (P < .05), and gastrocnemius (P < .05) muscles compared to untreated mdx mice. There were no significant differences in skeletal muscle function between treated and untreated groups. Chronic treatment with losartan decreases cardiac and skeletal muscle fibrosis and improves cardiac systolic function in dystrophin-deficient mdx mice. PMID:21304057

  10. Fetal skeletal muscle progenitors have regenerative capacity after intramuscular engraftment in dystrophin deficient mice.

    PubMed

    Sakai, Hiroshi; Sato, Takahiko; Sakurai, Hidetoshi; Yamamoto, Takuya; Hanaoka, Kazunori; Montarras, Didier; Sehara-Fujisawa, Atsuko

    2013-01-01

    Muscle satellite cells (SCs) are stem cells that reside in skeletal muscles and contribute to regeneration upon muscle injury. SCs arise from skeletal muscle progenitors expressing transcription factors Pax3 and/or Pax7 during embryogenesis in mice. However, it is unclear whether these fetal progenitors possess regenerative ability when transplanted in adult muscle. Here we address this question by investigating whether fetal skeletal muscle progenitors (FMPs) isolated from Pax3(GFP/+) embryos have the capacity to regenerate muscle after engraftment into Dystrophin-deficient mice, a model of Duchenne muscular dystrophy. The capacity of FMPs to engraft and enter the myogenic program in regenerating muscle was compared with that of SCs derived from adult Pax3(GFP/+) mice. Transplanted FMPs contributed to the reconstitution of damaged myofibers in Dystrophin-deficient mice. However, despite FMPs and SCs having similar myogenic ability in culture, the regenerative ability of FMPs was less than that of SCs in vivo. FMPs that had activated MyoD engrafted more efficiently to regenerate myofibers than MyoD-negative FMPs. Transcriptome and surface marker analyses of these cells suggest the importance of myogenic priming for the efficient myogenic engraftment. Our findings suggest the regenerative capability of FMPs in the context of muscle repair and cell therapy for degenerative muscle disease. PMID:23671652

  11. Fetal Skeletal Muscle Progenitors Have Regenerative Capacity after Intramuscular Engraftment in Dystrophin Deficient Mice

    PubMed Central

    Sakai, Hiroshi; Sato, Takahiko; Sakurai, Hidetoshi; Yamamoto, Takuya; Hanaoka, Kazunori; Montarras, Didier; Sehara-Fujisawa, Atsuko

    2013-01-01

    Muscle satellite cells (SCs) are stem cells that reside in skeletal muscles and contribute to regeneration upon muscle injury. SCs arise from skeletal muscle progenitors expressing transcription factors Pax3 and/or Pax7 during embryogenesis in mice. However, it is unclear whether these fetal progenitors possess regenerative ability when transplanted in adult muscle. Here we address this question by investigating whether fetal skeletal muscle progenitors (FMPs) isolated from Pax3GFP/+ embryos have the capacity to regenerate muscle after engraftment into Dystrophin-deficient mice, a model of Duchenne muscular dystrophy. The capacity of FMPs to engraft and enter the myogenic program in regenerating muscle was compared with that of SCs derived from adult Pax3GFP/+ mice. Transplanted FMPs contributed to the reconstitution of damaged myofibers in Dystrophin-deficient mice. However, despite FMPs and SCs having similar myogenic ability in culture, the regenerative ability of FMPs was less than that of SCs in vivo. FMPs that had activated MyoD engrafted more efficiently to regenerate myofibers than MyoD-negative FMPs. Transcriptome and surface marker analyses of these cells suggest the importance of myogenic priming for the efficient myogenic engraftment. Our findings suggest the regenerative capability of FMPs in the context of muscle repair and cell therapy for degenerative muscle disease. PMID:23671652

  12. Re-splicing of mature mRNA in cancer cells promotes activation of distant weak alternative splice sites

    PubMed Central

    Kameyama, Toshiki; Suzuki, Hitoshi; Mayeda, Akila

    2012-01-01

    Transcripts of the human tumor susceptibility gene 101 (TSG101) are aberrantly spliced in many cancers. A major aberrant splicing event on the TSG101 pre-mRNA involves joining of distant alternative 5′ and 3′ splice sites within exon 2 and exon 9, respectively, resulting in the extensive elimination of the mRNA. The estimated strengths of the alternative splice sites are much lower than those of authentic splice sites. We observed that the equivalent aberrant mRNA could be generated from an intron-less TSG101 gene expressed ectopically in breast cancer cells. Remarkably, we identified a pathway-specific endogenous lariat RNA consisting solely of exonic sequences, predicted to be generated by a re-splicing between exon 2 and exon 9 on the spliced mRNA. Our results provide evidence for a two-step splicing pathway in which the initial constitutive splicing removes all 14 authentic splice sites, thereby bringing the weak alternative splice sites into close proximity. We also demonstrate that aberrant multiple-exon skipping of the fragile histidine triad (FHIT) pre-mRNA in cancer cells occurs via re-splicing of spliced FHIT mRNA. The re-splicing of mature mRNA can potentially generate mutation-independent diversity in cancer transcriptomes. Conversely, a mechanism may exist in normal cells to prevent potentially deleterious mRNA re-splicing events. PMID:22675076

  13. Kinetic characterization of the first step of the ribozyme-catalyzed trans excision-splicing reaction.

    PubMed

    Dotson, P Patrick; Sinha, Joy; Testa, Stephen M

    2008-06-01

    Group I introns catalyze the self-splicing reaction, and their derived ribozymes are frequently used as model systems for the study of RNA folding and catalysis, as well as for the development of non-native catalytic reactions. Utilizing a group I intron-derived ribozyme from Pneumocystis carinii, we previously reported a non-native reaction termed trans excision-splicing (TES). In this reaction, an internal segment of RNA is excised from an RNA substrate, resulting in the covalent reattachment of the flanking regions. TES proceeds through two consecutive phosphotransesterification reactions, which are similar to the reaction steps of self-splicing. One key difference is that TES utilizes the 3'-terminal guanosine of the ribozyme as the first-step nucleophile, whereas self-splicing utilizes an exogenous guanosine. To further aid in our understanding of ribozyme reactions, a kinetic framework for the first reaction step (substrate cleavage) was established. The results demonstrate that the substrate binds to the ribozyme at a rate expected for simple helix formation. In addition, the rate constant for the first step of the TES reaction is more than one order of magnitude lower than the analogous step in self-splicing. Results also suggest that a conformational change, likely similar to that in self-splicing, exists between the two reaction steps of TES. Finally, multiple turnover is curtailed because dissociation of the cleavage product is slower than the rate of chemistry. PMID:18479464

  14. Genome-Wide Analysis of Alternative Splicing during Dendritic Cell Response to a Bacterial Challenge

    PubMed Central

    Rodrigues, Raquel; Grosso, Ana Rita; Moita, Luís

    2013-01-01

    The immune system relies on the plasticity of its components to produce appropriate responses to frequent environmental challenges. Dendritic cells (DCs) are critical initiators of innate immunity and orchestrate the later and more specific adaptive immunity. The generation of diversity in transcriptional programs is central for effective immune responses. Alternative splicing is widely considered a key generator of transcriptional and proteomic complexity, but its role has been rarely addressed systematically in immune cells. Here we used splicing-sensitive arrays to assess genome-wide gene- and exon-level expression profiles in human DCs in response to a bacterial challenge. We find widespread alternative splicing events and splicing factor transcriptional signatures induced by an E. coli challenge to human DCs. Alternative splicing acts in concert with transcriptional modulation, but these two mechanisms of gene regulation affect primarily distinct functional gene groups. Alternative splicing is likely to have an important role in DC immunobiology because it affects genes known to be involved in DC development, endocytosis, antigen presentation and cell cycle arrest. PMID:23613991

  15. The evolutionary landscape of intergenic trans-splicing events in insects

    PubMed Central

    Kong, Yimeng; Zhou, Hongxia; Yu, Yao; Chen, Longxian; Hao, Pei; Li, Xuan

    2015-01-01

    To explore the landscape of intergenic trans-splicing events and characterize their functions and evolutionary dynamics, we conduct a mega-data study of a phylogeny containing eight species across five orders of class Insecta, a model system spanning 400 million years of evolution. A total of 1,627 trans-splicing events involving 2,199 genes are identified, accounting for 1.58% of the total genes. Homology analysis reveals that mod(mdg4)-like trans-splicing is the only conserved event that is consistently observed in multiple species across two orders, which represents a unique case of functional diversification involving trans-splicing. Thus, evolutionarily its potential for generating proteins with novel function is not broadly utilized by insects. Furthermore, 146 non-mod trans-spliced transcripts are found to resemble canonical genes from different species. Trans-splicing preserving the function of ‘breakup' genes may serve as a general mechanism for relaxing the constraints on gene structure, with profound implications for the evolution of genes and genomes. PMID:26521696

  16. Identification of a Chloroplast Ribonucleoprotein Complex Containing Trans-splicing Factors, Intron RNA, and Novel Components*

    PubMed Central

    Jacobs, Jessica; Marx, Christina; Kock, Vera; Reifschneider, Olga; Fränzel, Benjamin; Krisp, Christoph; Wolters, Dirk; Kück, Ulrich

    2013-01-01

    Maturation of chloroplast psaA pre-mRNA from the green alga Chlamydomonas reinhardtii requires the trans-splicing of two split group II introns. Several nuclear-encoded trans-splicing factors are required for the correct processing of psaA mRNA. Among these is the recently identified Raa4 protein, which is involved in splicing of the tripartite intron 1 of the psaA precursor mRNA. Part of this tripartite group II intron is the chloroplast encoded tscA RNA, which is specifically bound by Raa4. Using Raa4 as bait in a combined tandem affinity purification and mass spectrometry approach, we identified core components of a multisubunit ribonucleoprotein complex, including three previously identified trans-splicing factors (Raa1, Raa3, and Rat2). We further detected tscA RNA in the purified protein complex, which seems to be specific for splicing of the tripartite group II intron. A yeast-two hybrid screen and co-immunoprecipitation identified chloroplast-localized Raa4-binding protein 1 (Rab1), which specifically binds tscA RNA from the tripartite psaA group II intron. The yeast-two hybrid system provides evidence in support of direct interactions between Rab1 and four trans-splicing factors. Our findings contribute to our knowledge of chloroplast multisubunit ribonucleoprotein complexes and are discussed in support of the generally accepted view that group II introns are the ancestors of the eukaryotic spliceosomal introns. PMID:23559604

  17. mRNA and microRNA transcriptomics analyses in a murine model of dystrophin loss and therapeutic restoration

    PubMed Central

    Roberts, Thomas C.; Blomberg, K. Emelie M.; Smith, C.I. Edvard; EL Andaloussi, Samir; Wood, Matthew J.A.

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a pediatric, X-linked, progressive muscle-wasting disorder caused by loss of function mutations affecting the gene encoding the dystrophin protein. While the primary genetic insult in DMD is well described, many details of the molecular and cellular pathologies that follow dystrophin loss are incompletely understood. To investigate gene expression in dystrophic muscle we have applied mRNA and microRNA (miRNA) microarray technology to the mdx mouse model of DMD. This study was designed to generate a complete description of gene expression changes associated with dystrophic pathology and the response to an experimental therapy which restores dystrophin protein function. These datasets have enabled (1) the determination of gene expression changes associated with dystrophic pathology, (2) identification of differentially expressed genes that are restored towards wild-type levels after therapeutic dystrophin rescue, (3) investigation of the correlation between mRNA and protein expression (determined by parallel mass spectrometry proteomics analysis), and (4) prediction of pathology associated miRNA-target interactions. Here we describe in detail how the data were generated including the basic analysis as contained in the manuscript published in Human Molecular Genetics with PMID 26385637. The data have been deposited in the Gene Expression Omnibus (GEO) with the accession number GSE64420. PMID:26981371

  18. mRNA and microRNA transcriptomics analyses in a murine model of dystrophin loss and therapeutic restoration.

    PubMed

    Roberts, Thomas C; Blomberg, K Emelie M; Smith, C I Edvard; El Andaloussi, Samir; Wood, Matthew J A

    2016-03-01

    Duchenne muscular dystrophy (DMD) is a pediatric, X-linked, progressive muscle-wasting disorder caused by loss of function mutations affecting the gene encoding the dystrophin protein. While the primary genetic insult in DMD is well described, many details of the molecular and cellular pathologies that follow dystrophin loss are incompletely understood. To investigate gene expression in dystrophic muscle we have applied mRNA and microRNA (miRNA) microarray technology to the mdx mouse model of DMD. This study was designed to generate a complete description of gene expression changes associated with dystrophic pathology and the response to an experimental therapy which restores dystrophin protein function. These datasets have enabled (1) the determination of gene expression changes associated with dystrophic pathology, (2) identification of differentially expressed genes that are restored towards wild-type levels after therapeutic dystrophin rescue, (3) investigation of the correlation between mRNA and protein expression (determined by parallel mass spectrometry proteomics analysis), and (4) prediction of pathology associated miRNA-target interactions. Here we describe in detail how the data were generated including the basic analysis as contained in the manuscript published in Human Molecular Genetics with PMID 26385637. The data have been deposited in the Gene Expression Omnibus (GEO) with the accession number GSE64420. PMID:26981371

  19. Rescue of a dystrophin-like protein by exon skipping normalizes synaptic plasticity in the hippocampus of the mdx mouse.

    PubMed

    Dallérac, Glenn; Perronnet, Caroline; Chagneau, Carine; Leblanc-Veyrac, Pascale; Samson-Desvignes, Nathalie; Peltekian, Elise; Danos, Olivier; Garcia, Luis; Laroche, Serge; Billard, Jean-Marie; Vaillend, Cyrille

    2011-09-01

    Duchenne muscular dystrophy (DMD) is caused by the absence of dystrophin, a protein that fulfills important functions in both muscle and brain. The mdx mouse model of DMD, which also lacks dystrophin, shows a marked reduction in γ-aminobutyric acid type A (GABA(A))-receptor clustering in central inhibitory synapses and enhanced long-term potentiation (LTP) at CA3-CA1 synapses of the hippocampus. We have recently shown that U7 small nuclear RNAs modified to encode antisense sequences and expressed from recombinant adeno-associated viral (rAAV) vectors are able to induce skipping of the mutated exon 23 and to rescue expression of a functional dystrophin-like product both in the muscle and nervous tissue in vivo. In the brain, this rescue was accompanied by restoration of both the size and number of hippocampal GABA(A)-receptor clustering. Here, we report that 25.2±8% of re-expression two months after intrahippocampal injection of rAAV reverses the abnormally enhanced LTP phenotype at CA3-CA1 synapses of mdx mice. These results suggests that dystrophin expression indirectly influences synaptic plasticity through modulation of GABA(A)-receptor clustering and that re-expression of the otherwise deficient protein in the adult can significantly alleviate alteration of neural functions in DMD. PMID:21624465

  20. Identification of alternative splicing regulators by RNA interference in Drosophila

    PubMed Central

    Park, Jung W.; Parisky, Katherine; Celotto, Alicia M.; Reenan, Robert A.; Graveley, Brenton R.

    2004-01-01

    Alternative splicing is thought to be regulated by nonspliceosomal RNA binding proteins that modulate the association of core components of the spliceosome with the pre-mRNA. Although the majority of metazoan genes encode pre-mRNAs that are alternatively spliced, remarkably few splicing regulators are currently known. Here, we used RNA interference to examine the role of >70% of the Drosophila RNA-binding proteins in regulating alternative splicing. We identified 47 proteins as splicing regulators, 26 of which have not previously been implicated in alternative splicing. Many of the regulators we identified are nonspliceosomal RNA-binding proteins. However, our screen unexpectedly revealed that altering the concentration of certain core components of the spliceosome specifically modulates alternative splicing. These results significantly expand the number of known splicing regulators and reveal an extraordinary richness in the mechanisms that regulate alternative splicing. PMID:15492211

  1. Splicing in action: assessing disease causing sequence changes

    PubMed Central

    Baralle, D; Baralle, M

    2005-01-01

    Variations in new splicing regulatory elements are difficult to identify exclusively by sequence inspection and may result in deleterious effects on precursor (pre) mRNA splicing. These mutations can result in either complete skipping of the exon, retention of the intron, or the introduction of a new splice site within an exon or intron. Sometimes mutations that do not disrupt or create a splice site activate pre-existing pseudo splice sites, consistent with the proposal that introns contain splicing inhibitory sequences. These variants can also affect the fine balance of isoforms produced by alternatively spliced exons and in consequence cause disease. Available genomic pathology data reveal that we are still partly ignorant of the basic mechanisms that underlie the pre-mRNA splicing process. The fact that human pathology can provide pointers to new modulatory elements of splicing should be exploited. PMID:16199547

  2. Does distance matter? Variations in alternative 3' splicing regulation.

    PubMed

    Akerman, Martin; Mandel-Gutfreund, Yael

    2007-01-01

    Alternative splicing constitutes a major mechanism creating protein diversity in humans. This diversity can result from the alternative skipping of entire exons or by alternative selection of the 5' or 3' splice sites that define the exon boundaries. In this study, we analyze the sequence and evolutionary characteristics of alternative 3' splice sites conserved between human and mouse genomes for distances ranging from 3 to 100 nucleotides. We show that alternative splicing events can be distinguished from constitutive splicing by a combination of properties which vary depending on the distance between the splice sites. Among the unique features of alternative 3' splice sites, we observed an unexpectedly high occurrence of events in which a polypyrimidine tract was found to overlap the upstream splice site. By applying a machine-learning approach, we show that we can successfully discriminate true alternative 3' splice sites from constitutive 3' splice sites. Finally, we propose that the unique features of the intron flanking alternative splice sites are indicative of a regulatory mechanism that is involved in splice site selection. We postulate that the process of splice site selection is influenced by the distance between the competitive splice sites. PMID:17704130

  3. Splicing therapy for neuromuscular disease.

    PubMed

    Douglas, Andrew G L; Wood, Matthew J A

    2013-09-01

    Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA) are two of the most common inherited neuromuscular diseases in humans. Both conditions are fatal and no clinically available treatments are able to significantly alter disease course in either case. However, by manipulation of pre-mRNA splicing using antisense oligonucleotides, defective transcripts from the DMD gene and from the SMN2 gene in SMA can be modified to once again produce protein and restore function. A large number of in vitro and in vivo studies have validated the applicability of this approach and an increasing number of preliminary clinical trials have either been completed or are under way. Several different oligonucleotide chemistries can be used for this purpose and various strategies are being developed to facilitate increased delivery efficiency and prolonged therapeutic effect. As these novel therapeutic compounds start to enter the clinical arena, attention must also be drawn to the question of how best to facilitate the clinical development of such personalised genetic therapies and how best to implement their provision. PMID:23631896

  4. Design of a Split Intein with Exceptional Protein Splicing Activity.

    PubMed

    Stevens, Adam J; Brown, Zachary Z; Shah, Neel H; Sekar, Giridhar; Cowburn, David; Muir, Tom W

    2016-02-24

    Protein trans-splicing (PTS) by split inteins has found widespread use in chemical biology and biotechnology. Herein, we describe the use of a consensus design approach to engineer a split intein with enhanced stability and activity that make it more robust than any known PTS system. Using batch mutagenesis, we first conduct a detailed analysis of the difference in splicing rates between the Npu (fast) and Ssp (slow) split inteins of the DnaE family and find that most impactful residues lie on the second shell of the protein, directly adjacent to the active site. These residues are then used to generate an alignment of 73 naturally occurring DnaE inteins that are predicted to be fast. The consensus sequence from this alignment (Cfa) demonstrates both rapid protein splicing and unprecedented thermal and chaotropic stability. Moreover, when fused to various proteins including antibody heavy chains, the N-terminal fragment of Cfa exhibits increased expression levels relative to other N-intein fusions. The durability and efficiency of Cfa should improve current intein based technologies and may provide a platform for the development of new protein chemistry techniques. PMID:26854538

  5. An Engineered Split Intein for Photoactivated Protein Trans-Splicing.

    PubMed

    Wong, Stanley; Mosabbir, Abdullah A; Truong, Kevin

    2015-01-01

    Protein splicing is mediated by inteins that auto-catalytically join two separated protein fragments with a peptide bond. Here we engineered a genetically encoded synthetic photoactivatable intein (named LOVInC), by using the light-sensitive LOV2 domain from Avena sativa as a switch to modulate the splicing activity of the split DnaE intein from Nostoc punctiforme. Periodic blue light illumination of LOVInC induced protein splicing activity in mammalian cells. To demonstrate the broad applicability of LOVInC, synthetic protein systems were engineered for the light-induced reassembly of several target proteins such as fluorescent protein markers, a dominant positive mutant of RhoA, caspase-7, and the genetically encoded Ca2+ indicator GCaMP2. Spatial precision of LOVInC was demonstrated by targeting activity to specific mammalian cells. Thus, LOVInC can serve as a general platform for engineering light-based control for modulating the activity of many different proteins. PMID:26317656

  6. Alternative Splicing Signatures in RNA-seq Data: Percent Spliced in (PSI).

    PubMed

    Schafer, Sebastian; Miao, Kui; Benson, Craig C; Heinig, Matthias; Cook, Stuart A; Hubner, Norbert

    2015-01-01

    Thousands of alternative exons are spliced out of messenger RNA to increase protein diversity. High-throughput sequencing of short cDNA fragments (RNA-seq) generates a genome-wide snapshot of these post-transcriptional processes. RNA-seq reads yield insights into the regulation of alternative splicing by revealing the usage of known or unknown splice sites as well as the expression level of exons. Constitutive exons are never covered by split alignments, whereas alternative exonic parts are located within highly expressed splicing junctions. The ratio between reads including or excluding exons, also known as percent spliced in index (PSI), indicates how efficiently sequences of interest are spliced into transcripts. This protocol describes a method to calculate the PSI without prior knowledge of splicing patterns. It provides a quantitative, global assessment of exon usage that can be integrated with other tools that identify differential isoform processing. Novel, complex splicing events along a genetic locus can be visualized in an exon-centric manner and compared across conditions. PMID:26439713

  7. An Alu-derived intronic splicing enhancer facilitates intronic processing and modulates aberrant splicing in ATM

    PubMed Central

    Pastor, Tibor; Talotti, Gabriele; Lewandowska, Marzena Anna; Pagani, Franco

    2009-01-01

    We have previously reported a natural GTAA deletion within an intronic splicing processing element (ISPE) of the ataxia telangiectasia mutated (ATM) gene that disrupts a non-canonical U1 snRNP interaction and activates the excision of the upstream portion of the intron. The resulting pre-mRNA splicing intermediate is then processed to a cryptic exon, whose aberrant inclusion in the final mRNA is responsible for ataxia telangiectasia. We show here that the last 40 bases of a downstream intronic antisense Alu repeat are required for the activation of the cryptic exon by the ISPE deletion. Evaluation of the pre-mRNA splicing intermediate by a hybrid minigene assay indicates that the identified intronic splicing enhancer represents a novel class of enhancers that facilitates processing of splicing intermediates possibly by recruiting U1 snRNP to defective donor sites. In the absence of this element, the splicing intermediate accumulates and is not further processed to generate the cryptic exon. Our results indicate that Alu-derived sequences can provide intronic splicing regulatory elements that facilitate pre-mRNA processing and potentially affect the severity of disease-causing splicing mutations. PMID:19773425

  8. Functional Analysis of Mutations in Exon 9 of NF1 Reveals the Presence of Several Elements Regulating Splicing

    PubMed Central

    Hernández-Imaz, Elisabete; Martín, Yolanda; de Conti, Laura; Melean, German; Valero, Ana; Baralle, Marco; Hernández-Chico, Concepción

    2015-01-01

    Neurofibromatosis type 1 (NF1) is one of the most common human hereditary disorders, predisposing individuals to the development of benign and malignant tumors in the nervous system, as well as other clinical manifestations. NF1 is caused by heterozygous mutations in the NF1 gene and around 25% of the pathogenic changes affect pre-mRNA splicing. Since the molecular mechanisms affected by these mutations are poorly understood, we have analyzed the splicing mutations identified in exon 9 of NF1, which is particularly prone to such changes, to better define the possible splicing regulatory elements. Using a minigene approach, we studied the effect of five splicing mutations in this exon described in patients. These highlighted three regulatory motifs within the exon. An in vivo splicing analysis of an extensive collection of changes generated in the minigene demonstrated that the CG motif at c.910-911 is critical for the recognition of exon 9. We also found that the GC motif at c.945-946 is involved in exon recognition through SRSF2 and that this motif is part of a Composite Exon Splicing Regulatory Element made up of physically overlapping enhancer and silencer elements. Finally, through an in vivo splicing analysis and in vitro binding assays, we demonstrated that the c.1007G>A mutation creates an Exonic Splicing Silencer element that binds the hnRNPA1 protein. The complexity of the splicing regulatory elements present in exon 9 is most likely responsible for the fact that mutations in this region represent 25% of all exonic changes that affect splicing in the NF1 gene. PMID:26509978

  9. Some sufficient conditions for persistency and permanency of two stages DNA splicing languages via Yusof-Goode approach

    NASA Astrophysics Data System (ADS)

    Mudaber, Mohammad Hassan; Yusof, Yuhani; Mohamad, Mohd Sham

    2014-07-01

    Splicing system that was first introduced by Head makes a connection between field of formal language theory and molecular biology. This system modeled the biological process of splitting and ligating on double stranded deoxyribonucleic acid (DNA) molecules under effect of restriction enzymes and appropriate ligase. In this paper, the concept of two stages DNA splicing languages is introduced. Some sufficient conditions for persistency and permanency of the above DNA splicing languages focusing on two rules and two initial strings will be investigated by usingYusof-Good (Y-G) approach.

  10. BRCA1 EXON 11, a CERES (composite regulatory element of splicing) element involved in splice regulation.

    PubMed

    Tammaro, Claudia; Raponi, Michela; Wilson, David I; Baralle, Diana

    2014-01-01

    Unclassified variants (UV) of BRCA1 can affect normal pre-mRNA splicing. Here, we investigate the UV c.693G>A, a "silent" change in BRCA1 exon 11, which we have found induces aberrant splicing in patient carriers and in vitro. Using a minigene assay, we show that the UV c.693G>A has a strong effect on the splicing isoform ratio of BRCA1. Systematic site-directed mutagenesis of the area surrounding the nucleotide position c.693G>A induced variable changes in the level of exon 11 inclusion/exclusion in the mRNA, pointing to the presence of a complex regulatory element with overlapping enhancer and silencer functions. Accordingly, protein binding analysis in the region detected several splicing regulatory factors involved, including SRSF1, SRSF6 and SRSF9, suggesting that this sequence represents a composite regulatory element of splicing (CERES). PMID:25056543

  11. RNA splicing. The human splicing code reveals new insights into the genetic determinants of disease.

    PubMed

    Xiong, Hui Y; Alipanahi, Babak; Lee, Leo J; Bretschneider, Hannes; Merico, Daniele; Yuen, Ryan K C; Hua, Yimin; Gueroussov, Serge; Najafabadi, Hamed S; Hughes, Timothy R; Morris, Quaid; Barash, Yoseph; Krainer, Adrian R; Jojic, Nebojsa; Scherer, Stephen W; Blencowe, Benjamin J; Frey, Brendan J

    2015-01-01

    To facilitate precision medicine and whole-genome annotation, we developed a machine-learning technique that scores how strongly genetic variants affect RNA splicing, whose alteration contributes to many diseases. Analysis of more than 650,000 intronic and exonic variants revealed widespread patterns of mutation-driven aberrant splicing. Intronic disease mutations that are more than 30 nucleotides from any splice site alter splicing nine times as often as common variants, and missense exonic disease mutations that have the least impact on protein function are five times as likely as others to alter splicing. We detected tens of thousands of disease-causing mutations, including those involved in cancers and spinal muscular atrophy. Examination of intronic and exonic variants found using whole-genome sequencing of individuals with autism revealed misspliced genes with neurodevelopmental phenotypes. Our approach provides evidence for causal variants and should enable new discoveries in precision medicine. PMID:25525159

  12. Bortezomib (PS-341) Treatment Decreases Inflammation and Partially Rescues the Expression of the Dystrophin-Glycoprotein Complex in GRMD Dogs

    PubMed Central

    Araujo, Karla P. C.; Bonuccelli, Gloria; Duarte, Caio N.; Gaiad, Thais P.; Moreira, Dayson F.; Feder, David; Belizario, José E.; Miglino, Maria A.; Lisanti, Michael P.; Ambrosio, Carlos E.

    2013-01-01

    Golden retriever muscular dystrophy (GRMD) is a genetic myopathy corresponding to Duchenne muscular dystrophy (DMD) in humans. Muscle atrophy is known to be associated with degradation of the dystrophin-glycoprotein complex (DGC) via the ubiquitin-proteasome pathway. In the present study, we investigated the effect of bortezomib treatment on the muscle fibers of GRMD dogs. Five GRMD dogs were examined; two were treated (TD- Treated dogs) with the proteasome inhibitor bortezomib, and three were control dogs (CD). Dogs were treated with bortezomib using the same treatment regimen used for multiple myeloma. Pharmacodynamics were evaluated by measuring the inhibition of 20S proteasome activity in whole blood after treatment and comparing it to that in CD. We performed immunohistochemical studies on muscle biopsy specimens to evaluate the rescue of dystrophin and dystrophin-associated proteins in the muscles of GRMD dogs treated with bortezomib. Skeletal tissue from TD had lower levels of connective tissue deposition and inflammatory cell infiltration than CD as determined by histology, collagen morphometry and ultrastructural analysis. The CD showed higher expression of phospho-NFκB and TGF-β1, suggesting a more pronounced activation of anti-apoptotic factors and inflammatory molecules and greater connective tissue deposition, respectively. Immunohistochemical analysis demonstrated that dystrophin was not present in the sarcoplasmic membrane of either group. However, bortezomib-TD showed higher expression of α- and β-dystroglycan, indicating an improved disease histopathology phenotype. Significant inhibition of 20S proteasome activity was observed 1 hour after bortezomib administration in the last cycle when the dose was higher. Proteasome inhibitors may thus improve the appearance of GRMD muscle fibers, lessen connective tissue deposition and reduce the infiltration of inflammatory cells. In addition, proteasome inhibitors may rescue some dystrophin

  13. Phase 2a Study of Ataluren-Mediated Dystrophin Production in Patients with Nonsense Mutation Duchenne Muscular Dystrophy

    PubMed Central

    Finkel, Richard S.; Flanigan, Kevin M.; Wong, Brenda; Bönnemann, Carsten; Sampson, Jacinda; Sweeney, H. Lee; Reha, Allen; Northcutt, Valerie J.; Elfring, Gary; Barth, Jay; Peltz, Stuart W.

    2013-01-01

    Background Approximately 13% of boys with Duchenne muscular dystrophy (DMD) have a nonsense mutation in the dystrophin gene, resulting in a premature stop codon in the corresponding mRNA and failure to generate a functional protein. Ataluren (PTC124) enables ribosomal readthrough of premature stop codons, leading to production of full-length, functional proteins. Methods This Phase 2a open-label, sequential dose-ranging trial recruited 38 boys with nonsense mutation DMD. The first cohort (n = 6) received ataluren three times per day at morning, midday, and evening doses of 4, 4, and 8 mg/kg; the second cohort (n = 20) was dosed at 10, 10, 20 mg/kg; and the third cohort (n = 12) was dosed at 20, 20, 40 mg/kg. Treatment duration was 28 days. Change in full-length dystrophin expression, as assessed by immunostaining in pre- and post-treatment muscle biopsy specimens, was the primary endpoint. Findings Twenty three of 38 (61%) subjects demonstrated increases in post-treatment dystrophin expression in a quantitative analysis assessing the ratio of dystrophin/spectrin. A qualitative analysis also showed positive changes in dystrophin expression. Expression was not associated with nonsense mutation type or exon location. Ataluren trough plasma concentrations active in the mdx mouse model were consistently achieved at the mid- and high- dose levels in participants. Ataluren was generally well tolerated. Interpretation Ataluren showed activity and safety in this short-term study, supporting evaluation of ataluren 10, 10, 20 mg/kg and 20, 20, 40 mg/kg in a Phase 2b, double-blind, long-term study in nonsense mutation DMD. Trial Registration ClinicalTrials.gov NCT00264888 PMID:24349052

  14. Vitamin D and alternative splicing of RNA

    PubMed Central

    Zhou, Rui; Chun, Rene F.; Lisse, Thomas S.; Garcia, Alejandro J.; Xu, Jianzhong; Adams, John S.; Hewison, Martin

    2014-01-01

    The active form of vitamin D (1α,25-dihydroxyvitamin D, 1,25(OH)2D) exerts its genomic effects via binding to a nuclear high-affinity vitamin D receptor (VDR). Recent deep sequencing analysis of VDR binding locations across the complete genome has significantly expanded our understanding of the actions of vitamin D and VDR on gene transcription. However, these studies have also promoted appreciation of the extra-transcriptional impact of vitamin D on gene expression. It is now clear that vitamin D interacts with the epigenome via effects on DNA methylation, histone acetylation, and microRNA generation to maintain normal biological functions. There is also increasing evidence that vitamin D can influence pre-mRNA constitutive splicing and alternative splicing, although the mechanism for this remains unclear. Pre-mRNA splicing has long been thought to be a post-transcription RNA processing event, but current data indicate that this occurs co-transcriptionally. Several steroid hormones have been recognized to coordinately control gene transcription and pre-mRNA splicing through the recruitment of nuclear receptor co-regulators that can both control gene transcription and splicing. The current review will discuss this concept with specific reference to vitamin D, and the potential role of heterogeneous nuclear ribonucleoprotein C (hnRNPC), a nuclear factor with an established function in RNA splicing. hnRNPC, has been shown to be involved in the VDR transcriptional complex as a vitamin D-response element-binding protein (VDRE-BP), and may act as a coupling factor linking VDR-directed gene transcription with RNA splicing. In this way hnRNPC may provide an additional mechanism for the fine-tuning of vitamin D-regulated target gene expression. PMID:25447737

  15. Tau exon 10 alternative splicing and tauopathies

    PubMed Central

    Liu, Fei; Gong, Cheng-Xin

    2008-01-01

    Abnormalities of microtubule-associated protein tau play a central role in neurofibrillary degeneration in several neurodegenerative disorders that collectively called tauopathies. Six isoforms of tau are expressed in adult human brain, which result from alternative splicing of pre-mRNA generated from a single tau gene. Alternative splicing of tau exon 10 results in tau isoforms containing either three or four microtubule-binding repeats (3R-tau and 4R-tau, respectively). Approximately equal levels of 3R-tau and 4R-tau are expressed in normal adult human brain, but the 3R-tau/4R-tau ratio is altered in the brains in several tauopathies. Discovery of silence mutations and intronic mutations of tau gene in some individuals with frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), which only disrupt tau exon 10 splicing but do not alter tau's primary sequence, demonstrates that dysregulation of tau exon 10 alternative splicing and consequently of 3R-tau/4R-tau balance is sufficient to cause neurodegeneration and dementia. Here, we review the gene structure, transcripts and protein isoforms of tau, followed by the regulation of exon 10 splicing that determines the expression of 3R-tau or 4R-tau. Finally, dysregulation of exon 10 splicing of tau in several tauopathies is discussed. Understanding the molecular mechanisms by which tau exon 10 splicing is regulated and how it is disrupted in tauopathies will provide new insight into the mechanisms of these tauopathies and help identify new therapeutic targets to treat these disorders. PMID:18616804

  16. The behavior of bonded doubler splices for composite sandwich panels

    NASA Technical Reports Server (NTRS)

    Zeller, T. A.; Weisahaar, T. A.

    1980-01-01

    The results of an investigation into the behavior of adhesively bonded doubler splices of two composite material sandwich panels are presented. The splices are studied from three approaches: analytical; numerical (finite elements); and experimental. Several parameters that characterize the splice are developed to determine their influence upon joint strength. These parameters are: doubler overlap length; core stiffness; laminate bending stiffness; the size of the gap between the spliced sandwich panels; and room and elevated temperatures. Similarities and contrasts between these splices and the physically similar single and double lap joints are discussed. The results of this investigation suggest several possible approaches to improving the strength of the sandwich splices.

  17. Gene and alternative splicing annotation with AIR

    PubMed Central

    Florea, Liliana; Di Francesco, Valentina; Miller, Jason; Turner, Russell; Yao, Alison; Harris, Michael; Walenz, Brian; Mobarry, Clark; Merkulov, Gennady V.; Charlab, Rosane; Dew, Ian; Deng, Zuoming; Istrail, Sorin; Li, Peter; Sutton, Granger

    2005-01-01

    Designing effective and accurate tools for identifying the functional and structural elements in a genome remains at the frontier of genome annotation owing to incompleteness and inaccuracy of the data, limitations in the computational models, and shifting paradigms in genomics, such as alternative splicing. We present a methodology for the automated annotation of genes and their alternatively spliced mRNA transcripts based on existing cDNA and protein sequence evidence from the same species or projected from a related species using syntenic mapping information. At the core of the method is the splice graph, a compact representation of a gene, its exons, introns, and alternatively spliced isoforms. The putative transcripts are enumerated from the graph and assigned confidence scores based on the strength of sequence evidence, and a subset of the high-scoring candidates are selected and promoted into the annotation. The method is highly selective, eliminating the unlikely candidates while retaining 98% of the high-quality mRNA evidence in well-formed transcripts, and produces annotation that is measurably more accurate than some evidence-based gene sets. The process is fast, accurate, and fully automated, and combines the traditionally distinct gene annotation and alternative splicing detection processes in a comprehensive and systematic way, thus considerably aiding in the ensuing manual curation efforts. PMID:15632090

  18. Phosphoregulation of Ire1 RNase splicing activity

    PubMed Central

    Prischi, Filippo; Nowak, Piotr R.; Carrara, Marta; Ali, Maruf M. U.

    2014-01-01

    Ire1 is activated in response to accumulation of misfolded proteins within the endoplasmic reticulum as part of the unfolded protein response (UPR). It is a unique enzyme, possessing both kinase and RNase activity that is required for specific splicing of Xbp1 mRNA leading to UPR activation. How phosphorylation impacts on the Ire1 splicing activity is unclear. In this study, we isolate distinct phosphorylated species of Ire1 and assess their effects on RNase splicing both in vitro and in vivo. We find that phosphorylation within the kinase activation loop significantly increases RNase splicing in vitro. Correspondingly, mutants of Ire1 that cannot be phosphorylated on the activation loop show decreased specific Xbp1 and promiscuous RNase splicing activity relative to wild-type Ire1 in cells. These data couple the kinase phosphorylation reaction to the activation state of the RNase, suggesting that phosphorylation of the activation loop is an important step in Ire1-mediated UPR activation. PMID:24704861

  19. The neuronal splicing factor Nova controls alternative splicing in N-type and P-type CaV2 calcium channels.

    PubMed

    Allen, Summer E; Darnell, Robert B; Lipscombe, Diane

    2010-01-01

    Many cellular processes are involved in optimizing protein function for specific neuronal tasks; here we focus on alternative pre-mRNA splicing. Alternative pre-mRNA splicing gives cells the capacity to modify and selectively re-balance their existing pool of transcripts in a coordinated way across multiple mRNAs, thereby effecting relatively rapid and relatively stable changes in protein activity. Here we report on and discuss the coordinated regulation of two sites of alternative splicing, e24a and e31a, in P-type CaV2.1 and N-type CaV2.2 channels. These two exons encode 4 and 2 amino acids, respectively, in the extracellular linker regions between transmembrane spanning segments S3 and S4 in domains III and IV of each CaV2 subunit. Recent genome-wide screens of splicing factor-RNA binding events by Darnell and colleagues show that Nova-2 promotes inclusion of e24a in CaV2.2 mRNAs in brain. We review these studies and show that a homologous e24a is present in theCaV2 .1 gene, Cacna1a, and that it is expressed in different regions of the nervous system. Nova-2 enhances inclusion of e24a but represses e31a inclusion in CaV2.1 and CaV2.2 mRNAs in brain. It is likely that coordinated alternative pre-mRNA splicing across related CaV2 genes by common splicing factors, allows neurons to orchestrate changes in synaptic protein function while maintaining a balanced and functioning system. PMID:21150296

  20. Early right ventricular fibrosis and reduction in biventricular cardiac reserve in the dystrophin-deficient mdx heart.

    PubMed

    Meyers, Tatyana A; Townsend, DeWayne

    2015-02-15

    Duchenne muscular dystrophy (DMD) is a progressive disease of striated muscle deterioration. Respiratory and cardiac muscle dysfunction are particularly clinically relevant because they result in the leading causes of death in DMD patients. Despite the clinical and physiological significance of these systems, little has been done to understand the cardiorespiratory interaction in DMD. We show here that prior to the onset of global cardiac dysfunction, dystrophin-deficient mdx mice have increased cardiac fibrosis with the right ventricle being particularly affected. Using a novel biventricular cardiac catheterization technique coupled with cardiac stress testing, we demonstrate that both the right and left ventricles have significant reductions in both systolic and diastolic function in response to dobutamine. Unstimulated cardiac function is relatively normal except for a significant reduction in the ventricular pressure transient duration compared with controls. These biventricular analyses also reveal the absence of a dobutamine-induced increase in isovolumic relaxation in the right ventricle of control hearts. Simultaneous assessment of biventricular pressure demonstrates a dobutamine-dependent enhancement of coupling between the ventricles in control mice, which is absent in mdx mice. Furthermore, studies probing the passive-extension properties of the left ventricle demonstrate that the mdx heart is significantly more compliant compared with age-matched C57BL/10 hearts, which have an age-dependent stiffening that is completely absent from dystrophic hearts. These new results indicate that right ventricular fibrosis is an early indicator of the development of dystrophic cardiomyopathy, suggesting a mechanism by which respiratory insufficiency may accelerate the development of heart failure in DMD. PMID:25485898

  1. Xenopus Pax-2 displays multiple splice forms during embryogenesis and pronephric kidney development.

    PubMed

    Heller, N; Brändli, A W

    1997-12-01

    Kidney organogenesis is initiated with the formation of the pronephric kidney and requires Pax-2 gene function. We report here the cloning and characterization of Pax-2 cDNAs from the frog Xenopus laevis, a model system suitable for the study of early kidney organogenesis. We show that expression of Xenopus Pax-2 (XPax-2) genes was confined to the nervous system, sensory organs, the visceral arches, and the developing excretory system. DNA sequencing of XPax-2 cDNAs isolated from head and pronephric kidney libraries revealed seven novel alternatively spliced Pax-2 isoforms. They all retain DNA-binding domains, but can differ significantly in their C termini with some isoforms containing a novel Pax-2 exon. We investigated the spectrum of XPax-2 splice events in pronephric kidneys, animal cap cultures and in whole embryos. Splicing of XPax-2 transcripts was found to be extensive and temporally regulated during Xenopus embryogenesis. Since all investigated tissues expressed essentially the full spectrum of XPax-2 splice variants, we conclude that splicing of XPax-2 transcripts does not occur in a tissue-specific manner. PMID:9486533

  2. Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes.

    PubMed

    Ebstein, F; Textoris-Taube, K; Keller, C; Golnik, R; Vigneron, N; Van den Eynde, B J; Schuler-Thurner, B; Schadendorf, D; Lorenz, F K M; Uckert, W; Urban, S; Lehmann, A; Albrecht-Koepke, N; Janek, K; Henklein, P; Niewienda, A; Kloetzel, P M; Mishto, M

    2016-01-01

    Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100(mel)47-52/40-42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100(mel)47-52/40-42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8(+) T cell response. Importantly, we demonstrate that different gp100(mel)-derived spliced epitopes are generated and presented to CD8(+) T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100(mel)-derived spliced epitopes trigger activation of CD8(+) T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes. PMID:27049119

  3. SeeSite: Characterizing Relationships between Splice Junctions and Splicing Enhancers.

    PubMed

    Lo, Christine; Kakaradov, Boyko; Lokshtanov, Daniel; Boucher, Christina

    2014-01-01

    RNA splicing is a cellular process driven by the interaction between numerous regulatory sequences and binding sites, however, such interactions have been primarily explored by laboratory methods since computational tools largely ignore the relationship between different splicing elements. Current computational methods identify either splice sites or other regulatory sequences, such as enhancers and silencers. We present a novel approach for characterizing co-occurring relationships between splice site motifs and splicing enhancers. Our approach relies on an efficient algorithm for approximately solving Consensus Sequence with Outliers , an NP-complete string clustering problem. In particular, we give an algorithm for this problem that outputs near-optimal solutions in polynomial time. To our knowledge, this is the first formulation and computational attempt for detecting co-occurring sequence elements in RNA sequence data. Further, we demonstrate that SeeSite is capable of showing that certain ESEs are preferentially associated with weaker splice sites, and that there exists a co-occurrence relationship with splice site motifs. PMID:26356335

  4. Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes

    PubMed Central

    Ebstein, F.; Textoris-Taube, K.; Keller, C.; Golnik, R.; Vigneron, N.; Van den Eynde, B. J.; Schuler-Thurner, B.; Schadendorf, D.; Lorenz, F. K. M.; Uckert, W.; Urban, S.; Lehmann, A.; Albrecht-Koepke, N.; Janek, K.; Henklein, P.; Niewienda, A.; Kloetzel, P. M.; Mishto, M.

    2016-01-01

    Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100mel47–52/40–42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100mel47–52/40–42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8+ T cell response. Importantly, we demonstrate that different gp100mel-derived spliced epitopes are generated and presented to CD8+ T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100mel-derived spliced epitopes trigger activation of CD8+ T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes. PMID:27049119

  5. IntSplice: prediction of the splicing consequences of intronic single-nucleotide variations in the human genome.

    PubMed

    Shibata, Akihide; Okuno, Tatsuya; Rahman, Mohammad Alinoor; Azuma, Yoshiteru; Takeda, Jun-Ichi; Masuda, Akio; Selcen, Duygu; Engel, Andrew G; Ohno, Kinji

    2016-07-01

    Precise spatiotemporal regulation of splicing is mediated by splicing cis-elements on pre-mRNA. Single-nucleotide variations (SNVs) affecting intronic cis-elements possibly compromise splicing, but no efficient tool has been available to identify them. Following an effect-size analysis of each intronic nucleotide on annotated alternative splicing, we extracted 105 parameters that could affect the strength of the splicing signals. However, we could not generate reliable support vector regression models to predict the percent-splice-in (PSI) scores for normal human tissues. Next, we generated support vector machine (SVM) models using 110 parameters to directly differentiate pathogenic SNVs in the Human Gene Mutation Database and normal SNVs in the dbSNP database, and we obtained models with a sensitivity of 0.800±0.041 (mean and s.d.) and a specificity of 0.849±0.021. Our IntSplice models were more discriminating than SVM models that we generated with Shapiro-Senapathy score and MaxEntScan::score3ss. We applied IntSplice to a naturally occurring and nine artificial intronic mutations in RAPSN causing congenital myasthenic syndrome. IntSplice correctly predicted the splicing consequences for nine of the ten mutants. We created a web service program, IntSplice (http://www.med.nagoya-u.ac.jp/neurogenetics/IntSplice) to predict splicing-affecting SNVs at intronic positions from -50 to -3. PMID:27009626

  6. Neuronal differentiation modulates the dystrophin Dp71d binding to the nuclear matrix

    SciTech Connect

    Rodriguez-Munoz, Rafael; Villarreal-Silva, Marcela; Gonzalez-Ramirez, Ricardo; Garcia-Sierra, Francisco; Mondragon, Monica; Mondragon, Ricardo; Cerna, Joel; Cisneros, Bulmaro

    2008-10-24

    The function of dystrophin Dp71 in neuronal cells remains unknown. To approach this issue, we have selected the PC12 neuronal cell line. These cells express both a Dp71f cytoplasmic variant and a Dp71d nuclear isoform. In this study, we demonstrated by electron and confocal microscopy analyses of in situ nuclear matrices and Western blotting evaluation of cell extracts that Dp71d associates with the nuclear matrix. Interestingly, this binding is modulated during NGF-induced neuronal differentiation of PC12 cells with a twofold increment in the differentiated cells, compared to control cells. Also, distribution of Dp71d along the periphery of the nuclear matrix observed in the undifferentiated cells is replaced by intense fluorescent foci localized in Center of the nucleoskeletal structure. In summary, we revealed that Dp71d is a dynamic component of nuclear matrix that might participate in the nuclear modeling occurring during neuronal differentiation.

  7. [Dystrophin gene expression in patients with Duchenne muscular dystrophy after myoblast transplantation].

    PubMed

    Shishkin, S S; Terekhov, S M; Krokhina, T B; Shakhovskaia, N I; Podobedova, A N; Linnaia, G F; Tarasov, V I; Ovchinnikov, V I; Krakhmaleva, I N; Zakharov, S F; Ershova, E S; Limborskaia, S A; Pogoda, T V; Zotikov, E A; Kut'ina, R M; Tarksh, M A; Sukhorukov, V S; Gerasimova, N L

    2001-08-01

    Based on originally designed technique of myoblast cultivation and in accordance with the approved by the Russian Ministry of Health "one muscle treatment" protocol of myoblast transplantation to the Duchenne muscular dystrophy patients, the first in Russia clinical trial of this gene correction method was carried out. Immonologically related myoblast cultures (30 to 90 million cells per patient) were injected after all preliminary procedures into tibialis anterior muscles of four boys selected from a group of volunteer recipients (Duchenne muscular dystrophy patients) based on the analysis of a number of surface antigens in donor-recipient pairs. The condition of the patients remained satisfactory during the whole period of post-transplantation follow-up (from 6 months to 1.5 years). Six months after myoblast transplantation the presence of donor DNA or dystrophin synthesis was demonstrated in muscle biopsies of three out of four patients. This result confirms efficacy and safety of the procedure used. PMID:11642111

  8. Hexose enhances oligonucleotide delivery and exon skipping in dystrophin-deficient mdx mice.

    PubMed

    Han, Gang; Gu, Ben; Cao, Limin; Gao, Xianjun; Wang, Qingsong; Seow, Yiqi; Zhang, Ning; Wood, Matthew J A; Yin, HaiFang

    2016-01-01

    Carbohydrate-based infusion solutions are widely used in the clinic. Here we show that co-administration of phosphorodiamidate morpholino oligomers (PMOs) with glucose enhances exon-skipping activity in Duchenne muscular dystrophy (DMD) mdx mice. We identify a glucose-fructose (GF) formulation that potentiates PMO activity, completely corrects aberrant Dmd transcripts, restores dystrophin levels in skeletal muscles and achieves functional rescue without detectable toxicity. This activity is attributed to enhancement of GF-mediated PMO uptake in the muscle. We demonstrate that PMO cellular uptake is energy dependent, and that ATP from GF metabolism contributes to enhanced cellular uptake of PMO in the muscle. Collectively, we show that GF potentiates PMO activity by replenishing cellular energy stores under energy-deficient conditions in mdx mice. Our findings provide mechanistic insight into hexose-mediated oligonucleotide delivery and have important implications for the development of DMD exon-skipping therapy. PMID:26964641

  9. Restriction Factors Against Recombinant Adeno-associated Virus Vectormediated Gene Transfer in Dystrophin-deficient Muscles.

    PubMed

    Dupont, Jean-Baptiste

    2016-01-01

    Despite the unprecedented beneficial effects of rAAV gene therapy in animal models of Duchenne muscular dystrophy (DMD), the need to inject large amounts of vector in vivo to improve phenotype raises obvious biosafety concerns. While rAAV vectors generally exhibit a good safety profile, specific pathological phenotypes such as those observed in dystrophin-deficient muscles may promote immunotoxic/genotoxic effects. Increasing the therapeutic index of rAAV in DMD muscles by reducing the effective dose could be a pivotal means of ensuring efficient clinical translation. This requires a comprehensive understanding of the rAAV transduction process, which is almost always studied in non-pathological tissues or in vitro. In this review, we focus on the molecular fate of rAAV after injection, and how the individual stages of transduction could be affected in the context of DMD. PMID:27121109

  10. Hexose enhances oligonucleotide delivery and exon skipping in dystrophin-deficient mdx mice

    PubMed Central

    Han, Gang; Gu, Ben; Cao, Limin; Gao, Xianjun; Wang, Qingsong; Seow, Yiqi; Zhang, Ning; Wood, Matthew J. A.; Yin, HaiFang

    2016-01-01

    Carbohydrate-based infusion solutions are widely used in the clinic. Here we show that co-administration of phosphorodiamidate morpholino oligomers (PMOs) with glucose enhances exon-skipping activity in Duchenne muscular dystrophy (DMD) mdx mice. We identify a glucose–fructose (GF) formulation that potentiates PMO activity, completely corrects aberrant Dmd transcripts, restores dystrophin levels in skeletal muscles and achieves functional rescue without detectable toxicity. This activity is attributed to enhancement of GF-mediated PMO uptake in the muscle. We demonstrate that PMO cellular uptake is energy dependent, and that ATP from GF metabolism contributes to enhanced cellular uptake of PMO in the muscle. Collectively, we show that GF potentiates PMO activity by replenishing cellular energy stores under energy-deficient conditions in mdx mice. Our findings provide mechanistic insight into hexose-mediated oligonucleotide delivery and have important implications for the development of DMD exon-skipping therapy. PMID:26964641

  11. Tau Splicing and the Intricacies of Dementia

    PubMed Central

    Andreadis, Athena

    2011-01-01

    Tau is a microtubule associated protein that fulfills several functions critical for neuronal formation and health. Tau discharges its functions by producing multiple isoforms via regulated alternative splicing. These isoforms modulate tau function in normal brain by altering the domains of the protein, thereby influencing its localization, conformation and post-translational modifications and hence its availability and affinity for microtubules and other ligands. Disturbances in tau expression result in disruption of the neuronal cytoskeleton and formation of tau structures (neurofibrillary tangles) found in brains of dementia sufferers. More specifically, aberrations in tau splicing regulation directly cause several neurodegenerative diseases which lead to dementia. In this review, I present our cumulative knowledge of tau splicing regulation in connection with neurodegeneration and also briefly go over the still-extensive list of questions that are connected to tau (dys)function. PMID:21604267

  12. Exon circularization requires canonical splice signals.

    PubMed

    Starke, Stefan; Jost, Isabelle; Rossbach, Oliver; Schneider, Tim; Schreiner, Silke; Hung, Lee-Hsueh; Bindereif, Albrecht

    2015-01-01

    Circular RNAs (circRNAs), an abundant class of noncoding RNAs in higher eukaryotes, are generated from pre-mRNAs by circularization of adjacent exons. Using a set of 15 circRNAs, we demonstrated their cell-type-specific expression and circular versus linear processing in mammalian cells. Northern blot analysis combined with RNase H cleavage conclusively proved a circular configuration for two examples, LPAR1 and HIPK3. To address the circularization mechanism, we analyzed the sequence requirements using minigenes derived from natural circRNAs. Both canonical splice sites are required for circularization, although they vary in flexibility and potential use of cryptic sites. Surprisingly, we found that no specific circRNA exon sequence is necessary and that potential flanking intron structures can modulate circularization efficiency. In combination with splice inhibitor assays, our results argue that the canonical spliceosomal machinery functions in circRNA biogenesis, constituting an alternative splicing mode. PMID:25543144

  13. Origin of Spliceosomal Introns and Alternative Splicing

    PubMed Central

    Irimia, Manuel; Roy, Scott William

    2014-01-01

    In this work we review the current knowledge on the prehistory, origins, and evolution of spliceosomal introns. First, we briefly outline the major features of the different types of introns, with particular emphasis on the nonspliceosomal self-splicing group II introns, which are widely thought to be the ancestors of spliceosomal introns. Next, we discuss the main scenarios proposed for the origin and proliferation of spliceosomal introns, an event intimately linked to eukaryogenesis. We then summarize the evidence that suggests that the last eukaryotic common ancestor (LECA) had remarkably high intron densities and many associated characteristics resembling modern intron-rich genomes. From this intron-rich LECA, the different eukaryotic lineages have taken very distinct evolutionary paths leading to profoundly diverged modern genome structures. Finally, we discuss the origins of alternative splicing and the qualitative differences in alternative splicing forms and functions across lineages. PMID:24890509

  14. Modeling of DNA single stage splicing language via Yusof-Goode approach: One string with two rules

    NASA Astrophysics Data System (ADS)

    Lim, Wen Li; Yusof, Yuhani; Mudaber, Mohammad Hassan

    2015-02-01

    Splicing system plays a pivotal role in attempts to recombine sets of double-stranded DNA molecules when acted by restriction enzymes and ligase. Traditional method of finding the result of DNA recombination through experiment is both time and money consuming. Hence, finding the number of patterns of DNA single stage splicing language through formalism of splicing system is a way to optimize the searching process. From the biological perspective, it predicts the number of types of molecules that will exist in the system under existence of restriction enzymes and ligase. In this paper, some theorems, corollaries and examples that lead to the predictions of single stage splicing languages involving one pattern string and two rules are presented via Yusof-Goode approach.

  15. Somatodendritic and excitatory postsynaptic distribution of neuron-type dystrophin isoform, Dp40, in hippocampal neurons

    SciTech Connect

    Fujimoto, Takahiro; Itoh, Kyoko Yaoi, Takeshi; Fushiki, Shinji

    2014-09-12

    Highlights: • Identification of dystrophin (Dp) shortest isoform, Dp40, is a neuron-type Dp. • Dp40 expression is temporally and differentially regulated in comparison to Dp71. • Somatodendritic and nuclear localization of Dp40. • Dp40 is localized to excitatory postsynapses. • Dp40 might play roles in dendritic and synaptic functions. - Abstract: The Duchenne muscular dystrophy (DMD) gene produces multiple dystrophin (Dp) products due to the presence of several promoters. We previously reported the existence of a novel short isoform of Dp, Dp40, in adult mouse brain. However, the exact biochemical expression profile and cytological distribution of the Dp40 protein remain unknown. In this study, we generated a polyclonal antibody against the NH{sub 2}-terminal region of the Dp40 and identified the expression profile of Dp40 in the mouse brain. Through an analysis using embryonic and postnatal mouse cerebrums, we found that Dp40 emerged from the early neonatal stages until adulthood, whereas Dp71, an another Dp short isoform, was highly detected in both prenatal and postnatal cerebrums. Intriguingly, relative expressions of Dp40 and Dp71 were prominent in cultured dissociated neurons and non-neuronal cells derived from mouse hippocampus, respectively. Furthermore, the immunocytological distribution of Dp40 was analyzed in dissociated cultured neurons, revealing that Dp40 is detected in the soma and its dendrites, but not in the axon. It is worthy to note that Dp40 is localized along the subplasmalemmal region of the dendritic shafts, as well as at excitatory postsynaptic sites. Thus, Dp40 was identified as a neuron-type Dp possibly involving dendritic and synaptic functions.

  16. Low-loss, robust fusion splicing of silica to chalcogenide fiber for integrated mid-infrared laser technology development.

    PubMed

    Thapa, Rajesh; Gattass, Rafael R; Nguyen, Vinh; Chin, Geoff; Gibson, Dan; Kim, Woohong; Shaw, L Brandon; Sanghera, Jasbinder S

    2015-11-01

    We demonstrate a low-loss, repeatable, and robust splice between single-mode silica fiber and single-mode chalcogenide (CHG) fiber. These splices are particularly difficult to create because of the significant difference in the two fibers' glass transition temperatures (∼1000°C) as well as the large difference in the coefficients of thermal expansion between the fibers (∼20×10(-6)/°C). With 90% light coupled through the silica-CHG fiber splice, predominantly in the fundamental circular-symmetric mode, into the core of the CHG fiber and with 0.5 dB of splice loss measured around the wavelength of 2.5 μm, after correcting only for the Fresnel loss, the silica-CHG splice offers excellent beam quality and coupling efficiency. The tensile strength of the splice is greater than 12 kpsi, and the laser damage threshold is greater than 2 W (CW) and was limited by the available laser pump power. We also utilized this splicing technique to demonstrate 2 to 4.5 μm ultrabroadband supercontinuum generation in a monolithic all-fiber system comprising a CHG fiber and a high peak power 2 μm pulsed Raman-shifted thulium fiber laser. This is a major development toward compact form factor commercial applications of soft-glass mid-IR fibers. PMID:26512522

  17. Dysregulation of splicing proteins in head and neck squamous cell carcinoma.

    PubMed

    Radhakrishnan, Aneesha; Nanjappa, Vishalakshi; Raja, Remya; Sathe, Gajanan; Chavan, Sandip; Nirujogi, Raja Sekhar; Patil, Arun H; Solanki, Hitendra; Renuse, Santosh; Sahasrabuddhe, Nandini A; Mathur, Premendu P; Prasad, T S Keshava; Kumar, Prashant; Califano, Joseph A; Sidransky, David; Pandey, Akhilesh; Gowda, Harsha; Chatterjee, Aditi

    2016-02-01

    ABSRTRACT Signaling plays an important role in regulating all cellular pathways. Altered signaling is one of the hallmarks of cancers. Phosphoproteomics enables interrogation of kinase mediated signaling pathways in biological systems. In cancers, this approach can be utilized to identify aberrantly activated pathways that potentially drive proliferation and tumorigenesis. To identify signaling alterations in head and neck squamous cell carcinoma (HNSCC), we carried out proteomic and phosphoproteomic analysis of HNSCC cell lines using a combination of tandem mass tag (TMT) labeling approach and titanium dioxide-based enrichment. We identified 4,920 phosphosites corresponding to 2,212 proteins in six HNSCC cell lines compared to a normal oral cell line. Our data indicated significant enrichment of proteins associated with splicing. We observed hyperphosphorylation of SRSF protein kinase 2 (SRPK2) and its downstream substrates in HNSCC cell lines. SRPK2 is a splicing kinase, known to phosphorylate serine/arginine (SR) rich domain proteins and regulate splicing process in eukaryotes. Although genome-wide studies have reported the contribution of alternative splicing events of several genes in the progression of cancer, the involvement of splicing kinases in HNSCC is not known. In this study, we studied the role of SRPK2 in HNSCC. Inhibition of SRPK2 resulted in significant decrease in colony forming and invasive ability in a panel of HNSCC cell lines. Our results indicate that phosphorylation of SRPK2 plays a crucial role in the regulation of splicing process in HNSCC and that splicing kinases can be developed as a new class of therapeutic target in HNSCC. PMID:26853621

  18. Depolarization and CaM kinase IV modulate NMDA receptor splicing through two essential RNA elements.

    PubMed

    Lee, Ji-Ann; Xing, Yi; Nguyen, David; Xie, Jiuyong; Lee, Christopher J; Black, Douglas L

    2007-02-01

    Alternative splicing controls the activity of many proteins important for neuronal excitation, but the signal-transduction pathways that affect spliced isoform expression are not well understood. One particularly interesting system of alternative splicing is exon 21 (E21) of the NMDA receptor 1 (NMDAR1 E21), which controls the trafficking of NMDA receptors to the plasma membrane and is repressed by Ca(++)/calmodulin-dependent protein kinase (CaMK) IV signaling. Here, we characterize the splicing of NMDAR1 E21. We find that E21 splicing is reversibly repressed by neuronal depolarization, and we identify two RNA elements within the exon that function together to mediate the inducible repression. One of these exonic elements is similar to an intronic CaMK IV-responsive RNA element (CaRRE) originally identified in the 3' splice site of the BK channel STREX exon, but not previously observed within an exon. The other element is a new RNA motif. Introduction of either of these two motifs, called CaRRE type 1 and CaRRE type 2, into a heterologous constitutive exon can confer CaMK IV-dependent repression on the new exon. Thus, either exonic CaRRE can be sufficient for CaMK IV-induced repression. Single nucleotide scanning mutagenesis defined consensus sequences for these two CaRRE motifs. A genome-wide motif search and subsequent RT-PCR validation identified a group of depolarization-regulated alternative exons carrying CaRRE consensus sequences. Many of these exons are likely to alter neuronal function. Thus, these two RNA elements define a group of co-regulated splicing events that respond to a common stimulus in neurons to alter their activity. PMID:17298178

  19. SPA: A Probabilistic Algorithm for Spliced Alignment

    PubMed Central

    van Nimwegen, Erik; Paul, Nicodeme; Sheridan, Robert; Zavolan, Mihaela

    2006-01-01

    Recent large-scale cDNA sequencing efforts show that elaborate patterns of splice variation are responsible for much of the proteome diversity in higher eukaryotes. To obtain an accurate account of the repertoire of splice variants, and to gain insight into the mechanisms of alternative splicing, it is essential that cDNAs are very accurately mapped to their respective genomes. Currently available algorithms for cDNA-to-genome alignment do not reach the necessary level of accuracy because they use ad hoc scoring models that cannot correctly trade off the likelihoods of various sequencing errors against the probabilities of different gene structures. Here we develop a Bayesian probabilistic approach to cDNA-to-genome alignment. Gene structures are assigned prior probabilities based on the lengths of their introns and exons, and based on the sequences at their splice boundaries. A likelihood model for sequencing errors takes into account the rates at which misincorporation, as well as insertions and deletions of different lengths, occurs during sequencing. The parameters of both the prior and likelihood model can be automatically estimated from a set of cDNAs, thus enabling our method to adapt itself to different organisms and experimental procedures. We implemented our method in a fast cDNA-to-genome alignment program, SPA, and applied it to the FANTOM3 dataset of over 100,000 full-length mouse cDNAs and a dataset of over 20,000 full-length human cDNAs. Comparison with the results of four other mapping programs shows that SPA produces alignments of significantly higher quality. In particular, the quality of the SPA alignments near splice boundaries and SPA's mapping of the 5′ and 3′ ends of the cDNAs are highly improved, allowing for more accurate identification of transcript starts and ends, and accurate identification of subtle splice variations. Finally, our splice boundary analysis on the human dataset suggests the existence of a novel non-canonical splice

  20. Possible influences on the expression of X chromosome-linked dystrophin abnormalities by heterozygosity for autosomal recessive Fukuyama congenital muscular dystrophy.

    PubMed Central

    Beggs, A H; Neumann, P E; Arahata, K; Arikawa, E; Nonaka, I; Anderson, M S; Kunkel, L M

    1992-01-01

    Abnormalities of dystrophin, a cytoskeletal protein of muscle and nerve, are generally considered specific for Duchenne and Becker muscular dystrophy. However, several patients have recently been identified with dystrophin deficiency who, before dystrophin testing, were considered to have Fukuyama congenital muscular dystrophy (FCMD) on the basis of clinical findings. Epidemiologic data suggest that only 1/3500 males with autosomal recessive FCMD should have abnormal dystrophin. To explain the observation of 3/23 FCMD males with abnormal dystrophin, we propose that dystrophin and the FCMD gene product interact and that the earlier onset and greater severity of these patients' phenotype (relative to Duchenne muscular dystrophy) are due to their being heterozygous for the FCMD mutation in addition to being hemizygous for Duchenne muscular dystrophy, a genotype that is predicted to occur in 1/175,000 Japanese males. This model may help explain the genetic basis for some of the clinical and pathological variability seen among patients with FCMD, and it has potential implications for understanding the inheritance of other autosomal recessive disorders in general. For example, sex ratios for rare autosomal recessive disorders caused by mutations in proteins that interact with X chromosome-linked gene products may display predictable deviation from 1:1. Images PMID:1731332

  1. Possible influences on the expression of X chromosome-linked dystrophin abnormalities by heterozygosity for autosomal recessive Fukuyama congenital muscular dystrophy

    SciTech Connect

    Beggs, A.H.; Neumann, P.E.; Anderson, M.S.; Kunkel, L.M. ); Arahata, Kiichi; Arikawa, Eri; Nonaka, Ikuya )

    1992-01-15

    Abnormalities of dystrophin, a cytoskeletal protein of muscle and nerve, are generally considered specific for Duchenne and Becker muscular dystrophy. However, several patients have recently been identified with dystrophin deficiency who, before dystrophin testing, were considered to have Fukuyama congenital muscular dystrophy (FCMD) on the basis of clinical findings. Epidemiologic data suggest that only 1/3,500 males with autosomal recessive FCMD should have abnormal dystrophin. To explain the observation of 3/23 FCMD males with abnormal dystrophin, the authors propose that dystrophin and the FCMD gene product interact and that the earlier onset and greater severity of these patients' phenotype (relative to Duchenne muscular dystrophy) are due to their being heterozygous for the FCMD mutation in addition to being hemizygous for Duchenne muscular dystrophy, a genotype that is predicted to occur in 1/175,000 Japanese males. This model may help explain the genetic basis for some of the clinical and pathological variability seen among patients with FCMD, and it has potential implications for understanding the inheritance of other autosomal recessive disorders in general. For example, sex ratios for rare autosomal recessive disorders caused by mutations in proteins that interact with X chromosome-linked gene products may display predictable deviation from 1:1.

  2. Pathological impact of SMN2 mis-splicing in adult SMA mice

    PubMed Central

    Sahashi, Kentaro; Ling, Karen K Y; Hua, Yimin; Wilkinson, John Erby; Nomakuchi, Tomoki; Rigo, Frank; Hung, Gene; Xu, David; Jiang, Ya-Ping; Lin, Richard Z; Ko, Chien-Ping; Bennett, C Frank; Krainer, Adrian R

    2013-01-01

    Loss-of-function mutations in SMN1 cause spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. The related SMN2 gene expresses suboptimal levels of functional SMN protein, due to a splicing defect. Many SMA patients reach adulthood, and there is also adult-onset (type IV) SMA. There is currently no animal model for adult-onset SMA, and the tissue-specific pathogenesis of post-developmental SMN deficiency remains elusive. Here, we use an antisense oligonucleotide (ASO) to exacerbate SMN2 mis-splicing. Intracerebroventricular ASO injection in adult SMN2-transgenic mice phenocopies key aspects of adult-onset SMA, including delayed-onset motor dysfunction and relevant histopathological features. SMN2 mis-splicing increases during late-stage disease, likely accelerating disease progression. Systemic ASO injection in adult mice causes peripheral SMN2 mis-splicing and affects prognosis, eliciting marked liver and heart pathologies, with decreased IGF1 levels. ASO dose–response and time-course studies suggest that only moderate SMN levels are required in the adult central nervous system, and treatment with a splicing-correcting ASO shows a broad therapeutic time window. We describe distinctive pathological features of adult-onset and early-onset SMA. PMID:24014320

  3. 30 CFR 77.602 - Permanent splicing of trailing cables.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Permanent splicing of trailing cables. 77.602... COAL MINES Trailing Cables § 77.602 Permanent splicing of trailing cables. When permanent splices in trailing cables are made, they shall be: (a) Mechanically strong with adequate electrical conductivity;...

  4. 46 CFR 111.60-19 - Cable splices.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... with section 25.11 of IEEE 45-2002 (incorporated by reference; see 46 CFR 110.10-1). ... 46 Shipping 4 2012-10-01 2012-10-01 false Cable splices. 111.60-19 Section 111.60-19 Shipping... REQUIREMENTS Wiring Materials and Methods § 111.60-19 Cable splices. (a) A cable must not be spliced in...

  5. 30 CFR 75.603 - Temporary splice of trailing cable.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Temporary splice of trailing cable. 75.603... SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.603 Temporary splice of trailing cable. One temporary splice may be made in any trailing cable. Such trailing cable...

  6. 30 CFR 75.603 - Temporary splice of trailing cable.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Temporary splice of trailing cable. 75.603... SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.603 Temporary splice of trailing cable. One temporary splice may be made in any trailing cable. Such trailing cable...

  7. 30 CFR 75.604 - Permanent splicing of trailing cables.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Permanent splicing of trailing cables. 75.604... SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.604 Permanent splicing of trailing cables. When permanent splices in trailing cables are made, they shall be:...

  8. 30 CFR 75.604 - Permanent splicing of trailing cables.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Permanent splicing of trailing cables. 75.604... SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.604 Permanent splicing of trailing cables. When permanent splices in trailing cables are made, they shall be:...

  9. 30 CFR 77.602 - Permanent splicing of trailing cables.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Permanent splicing of trailing cables. 77.602... COAL MINES Trailing Cables § 77.602 Permanent splicing of trailing cables. When permanent splices in trailing cables are made, they shall be: (a) Mechanically strong with adequate electrical conductivity;...

  10. 30 CFR 75.603 - Temporary splice of trailing cable.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Temporary splice of trailing cable. 75.603... SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.603 Temporary splice of trailing cable. One temporary splice may be made in any trailing cable. Such trailing cable...

  11. 30 CFR 77.602 - Permanent splicing of trailing cables.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Permanent splicing of trailing cables. 77.602... COAL MINES Trailing Cables § 77.602 Permanent splicing of trailing cables. When permanent splices in trailing cables are made, they shall be: (a) Mechanically strong with adequate electrical conductivity;...

  12. 46 CFR 111.60-19 - Cable splices.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... with section 25.11 of IEEE 45-2002 (incorporated by reference; see 46 CFR 110.10-1). ... 46 Shipping 4 2013-10-01 2013-10-01 false Cable splices. 111.60-19 Section 111.60-19 Shipping... REQUIREMENTS Wiring Materials and Methods § 111.60-19 Cable splices. (a) A cable must not be spliced in...

  13. 30 CFR 75.604 - Permanent splicing of trailing cables.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Permanent splicing of trailing cables. 75.604... SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.604 Permanent splicing of trailing cables. When permanent splices in trailing cables are made, they shall be:...

  14. 46 CFR 111.60-19 - Cable splices.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... with section 25.11 of IEEE 45-2002 (incorporated by reference; see 46 CFR 110.10-1). ... 46 Shipping 4 2011-10-01 2011-10-01 false Cable splices. 111.60-19 Section 111.60-19 Shipping... REQUIREMENTS Wiring Materials and Methods § 111.60-19 Cable splices. (a) A cable must not be spliced in...

  15. 30 CFR 75.604 - Permanent splicing of trailing cables.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Permanent splicing of trailing cables. 75.604... SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.604 Permanent splicing of trailing cables. When permanent splices in trailing cables are made, they shall be:...

  16. 30 CFR 77.602 - Permanent splicing of trailing cables.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Permanent splicing of trailing cables. 77.602... COAL MINES Trailing Cables § 77.602 Permanent splicing of trailing cables. When permanent splices in trailing cables are made, they shall be: (a) Mechanically strong with adequate electrical conductivity;...

  17. 30 CFR 75.603 - Temporary splice of trailing cable.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Temporary splice of trailing cable. 75.603... SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.603 Temporary splice of trailing cable. One temporary splice may be made in any trailing cable. Such trailing cable...

  18. The Effective Lifetime of ACSR Full Tension Splice Connector Operated at Higher Temperature

    SciTech Connect

    Wang, Jy-An John; Lara-Curzio, Edgar; King Jr, Thomas J; Graziano, Joe; Chan, John; Goodwin, Tip

    2009-01-01

    This paper is to address the issues related to integrity of ACSR full tension splice connectors operated at high temperatures. A protocol of integrating analytical and experimental approaches to evaluate the integrity of a full tension single-stage splice connector (SSC) assembly during service at high operating temperature was developed. Based on the developed protocol the effective lifetime evaluation was demonstrated with ACSR Drake conductor SSC systems. The investigation indicates that thermal cycling temperature and frequency, conductor cable tension loading, and the compressive residual stress field within a SSC system have significant impact on the SSC integrity and the associated effective lifetime.

  19. Decoding of exon splicing patterns in the human RUNX1-RUNX1T1 fusion gene.

    PubMed

    Grinev, Vasily V; Migas, Alexandr A; Kirsanava, Aksana D; Mishkova, Olga A; Siomava, Natalia; Ramanouskaya, Tatiana V; Vaitsiankova, Alina V; Ilyushonak, Ilia M; Nazarov, Petr V; Vallar, Laurent; Aleinikova, Olga V

    2015-11-01

    The t(8;21) translocation is the most widespread genetic defect found in human acute myeloid leukemia. This translocation results in the RUNX1-RUNX1T1 fusion gene that produces a wide variety of alternative transcripts and influences the course of the disease. The rules of combinatorics and splicing of exons in the RUNX1-RUNX1T1 transcripts are not known. To address this issue, we developed an exon graph model of the fusion gene organization and evaluated its local exon combinatorics by the exon combinatorial index (ECI). Here we show that the local exon combinatorics of the RUNX1-RUNX1T1 gene follows a power-law behavior and (i) the vast majority of exons has a low ECI, (ii) only a small part is represented by "exons-hubs" of splicing with very high ECI values, and (iii) it is scale-free and very sensitive to targeted skipping of "exons-hubs". Stochasticity of the splicing machinery and preferred usage of exons in alternative splicing can explain such behavior of the system. Stochasticity may explain up to 12% of the ECI variance and results in a number of non-coding and unproductive transcripts that can be considered as a noise. Half-life of these transcripts is increased due to the deregulation of some key genes of the nonsense-mediated decay system in leukemia cells. On the other hand, preferred usage of exons may explain up to 75% of the ECI variability. Our analysis revealed a set of splicing-related cis-regulatory motifs that can explain "attractiveness" of exons in alternative splicing but only when they are considered together. Cis-regulatory motifs are guides for splicing trans-factors and we observed a leukemia-specific profile of expression of the splicing genes in t(8;21)-positive blasts. Altogether, our results show that alternative splicing of the RUNX1-RUNX1T1 transcripts follows strict rules and that the power-law component of the fusion gene organization confers a high flexibility to this process. PMID:26320575

  20. Inhibition of Splicing but not Cleavage at the 5' Splice Site by Truncating Human β -globin Pre-mRNA

    NASA Astrophysics Data System (ADS)

    Furdon, Paul J.; Kole, Ryszard

    1986-02-01

    Human β -globin mRNAs truncated in the second exon or in the first intron have been processed in vitro in a HeLa cell nuclear extract. Transcripts containing a fragment of the second exon as short as 53 nucleotides are efficiently spliced, whereas transcripts truncated 24 or 14 nucleotides downstream from the 3' splice site are spliced inefficiently, if at all. All of these transcripts, however, are efficiently and accurately cleaved at the 5' splice site. In contrast, RNA truncated in the first intron, 54 nucleotides upstream from the 3' splice site, is not processed at all. These findings suggest that cleavage at the 5' splice site and subsequent splicing steps--i.e., cleavage at the 3' splice site and exon ligation--need not be coupled. Anti-Sm serum inhibits the complete splicing reaction and cleavage at the 5' splice site, suggesting involvement of certain ribonucleoprotein particles in the cleavage reaction. ATP and Mg2+ are required for cleavage at the 5' splice site at concentrations similar to those for the complete splicing reaction.

  1. Global genome splicing analysis reveals an increased number of alternatively spliced genes with aging.

    PubMed

    Rodríguez, Sofía A; Grochová, Diana; McKenna, Tomás; Borate, Bhavesh; Trivedi, Niraj S; Erdos, Michael R; Eriksson, Maria

    2016-04-01

    Alternative splicing (AS) is a key regulatory mechanism for the development of different tissues; however, not much is known about changes to alternative splicing during aging. Splicing events may become more frequent and widespread genome-wide as tissues age and the splicing machinery stringency decreases. Using skin, skeletal muscle, bone, thymus, and white adipose tissue from wild-type C57BL6/J male mice (4 and 18 months old), we examined the effect of age on splicing by AS analysis of the differential exon usage of the genome. The results identified a considerable number of AS genes in skeletal muscle, thymus, bone, and white adipose tissue between the different age groups (ranging from 27 to 246 AS genes corresponding to 0.3-3.2% of the total number of genes analyzed). For skin, skeletal muscle, and bone, we included a later age group (28 months old) that showed that the number of alternatively spliced genes increased with age in all three tissues (P < 0.01). Analysis of alternatively spliced genes across all tissues by gene ontology and pathway analysis identified 158 genes involved in RNA processing. Additional analysis of AS in a mouse model for the premature aging disease Hutchinson-Gilford progeria syndrome was performed. The results show that expression of the mutant protein, progerin, is associated with an impaired developmental splicing. As progerin accumulates, the number of genes with AS increases compared to in wild-type skin. Our results indicate the existence of a mechanism for increased AS during aging in several tissues, emphasizing that AS has a more important role in the aging process than previously known. PMID:26685868

  2. The HP1 homolog Rhino anchors a nuclear complex that suppresses piRNA precursor splicing

    PubMed Central

    Zhang, Zhao; Wang, Jie; Schultz, Nadine; Zhang, Fan; Parhad, Swapnil S.; Tu, Shikui; Vreven, Thom; Zamore, Phillip D.; Weng, Zhiping; Theurkauf, William E.

    2014-01-01

    Summary piRNAs guide an adaptive genome defense system that silences transposons during germline development. The Drosophila HP1 homolog Rhino is required for germline piRNA production. We show that Rhino binds specifically to the heterochromatic clusters that produce piRNA precursors, and that binding directly correlates with piRNA production. Rhino co-localizes to germline nuclear foci with Rai1/DXO related protein Cuff and the DEAD box protein UAP56, which are also required for germline piRNA production. RNA sequencing indicates that most cluster transcripts are not spliced, and that rhino, cuff and uap56 mutations increase expression of spliced cluster transcripts over 100 fold. LacI∷Rhino fusion protein binding suppresses splicing of a reporter transgene, and is sufficient to trigger piRNA production from a trans combination of sense and antisense reporters. We therefore propose that Rhino anchors a nuclear complex that suppresses cluster transcript splicing, and speculate that stalled splicing differentiates piRNA precursors from mRNAs. PMID:24906152

  3. Production of the 2400 kb Duchenne muscular dystrophy (DMD) gene transcript; transcription time and cotranscriptional splicing

    SciTech Connect

    Tennyson, C.N.; Worton, R.G.

    1994-09-01

    The largest known gene in any organism is the human DMD gene which has 79 exons that span 2400 kb. The extreme nature of the DMD gene raises questions concerning the time required for transcription and whether splicing begins before transcription is complete. DMD gene transcription is induced as cultured human myoblasts differentiate to form multinucleated myotubes, providing a system for studying the kinetics of transcription and splicing. Using quantitative RT-PCR, transcript accumulation was monitored from four different regions within the gene following induction of expression. By comparing the accumulation of transcripts from the 5{prime} and 3{prime} ends of the gene we have shown that approximately 12 hours are required to transcribe 1770 kb of the gene, extrapolating to a time of 16 hours for the transcription unit expressed in muscle. Comparison of accumulation profiles for spliced and total transcript demonstrated that transcripts are spliced at the 5{prime} end before transcription is complete, providing strong evidence for cotranscriptional splicing of DMD gene transcripts. Finally, the rate of transcript accumulation was reduced at the 3{prime} end of the gene relative to the 5{prime} end, perhaps due to premature termination of transcription complexes as they traverse this enormous transcription unit. The lag between transcription initiation and the appearance of complete transcripts could be important in limiting transcript production in dividing cells and to the timing of mRNA appearance in differentiating muscle.

  4. Histone methylation, alternative splicing and neuronal differentiation.

    PubMed

    Fiszbein, Ana; Kornblihtt, Alberto R

    2016-01-01

    Alternative splicing, as well as chromatin structure, greatly contributes to specific transcriptional programs that promote neuronal differentiation. The activity of G9a, the enzyme responsible for mono- and di-methylation of lysine 9 on histone H3 (H3K9me1 and H3K9me2) in mammalian euchromatin, has been widely implicated in the differentiation of a variety of cell types and tissues. In a recent work from our group (Fiszbein et al., 2016) we have shown that alternative splicing of G9a regulates its nuclear localization and, therefore, the efficiency of H3K9 methylation, which promotes neuronal differentiation. We discuss here our results in the light of a report from other group (Laurent et al. 2015) demonstrating a key role for the alternative splicing of the histone demethylase LSD1 in controlling specific gene expression in neurons. All together, these results illustrate the importance of alternative splicing in the generation of a proper equilibrium between methylation and demethylation of histones for the regulation of neuron-specific transcriptional programs. PMID:27606339

  5. Alternative-splicing-mediated gene expression

    NASA Astrophysics Data System (ADS)

    Wang, Qianliang; Zhou, Tianshou

    2014-01-01

    Alternative splicing (AS) is a fundamental process during gene expression and has been found to be ubiquitous in eukaryotes. However, how AS impacts gene expression levels both quantitatively and qualitatively remains to be fully explored. Here, we analyze two common models of gene expression, each incorporating a simple splice mechanism that a pre-mRNA is spliced into two mature mRNA isoforms in a probabilistic manner. In the constitutive expression case, we show that the steady-state molecular numbers of two mature mRNA isoforms follow mutually independent Poisson distributions. In the bursting expression case, we demonstrate that the tail decay of the steady-state distribution for both mature mRNA isoforms that in general are not mutually independent can be characterized by the product of mean burst size and splicing probability. In both cases, we find that AS can efficiently modulate both the variability (measured by variance) and the noise level of the total mature mRNA, and in particular, the latter is always lower than the noise level of the pre-mRNA, implying that AS always reduces the noise. These results altogether reveal that AS is a mechanism of efficiently controlling the gene expression noise.

  6. RNA structure in splicing: An evolutionary perspective.

    PubMed

    Lin, Chien-Ling; Taggart, Allison J; Fairbrother, William G

    2016-09-01

    Pre-mRNA splicing is a key post-transcriptional regulation process in which introns are excised and exons are ligated together. A novel class of structured intron was recently discovered in fish. Simple expansions of complementary AC and GT dimers at opposite boundaries of an intron were found to form a bridging structure, thereby enforcing correct splice site pairing across the intron. In some fish introns, the RNA structures are strong enough to bypass the need of regulatory protein factors for splicing. Here, we discuss the prevalence and potential functions of highly structured introns. In humans, structured introns usually arise through the co-occurrence of C and G-rich repeats at intron boundaries. We explore the potentially instructive example of the HLA receptor genes. In HLA pre-mRNA, structured introns flank the exons that encode the highly polymorphic β sheet cleft, making the processing of the transcript robust to variants that disrupt splicing factor binding. While selective forces that have shaped HLA receptor are fairly atypical, numerous other highly polymorphic genes that encode receptors contain structured introns. Finally, we discuss how the elevated mutation rate associated with the simple repeats that often compose structured intron can make structured introns themselves rapidly evolving elements. PMID:27454491

  7. RNA splicing factors as oncoproteins and tumour suppressors.

    PubMed

    Dvinge, Heidi; Kim, Eunhee; Abdel-Wahab, Omar; Bradley, Robert K

    2016-07-01

    The recent genomic characterization of cancers has revealed recurrent somatic point mutations and copy number changes affecting genes encoding RNA splicing factors. Initial studies of these 'spliceosomal mutations' suggest that the proteins bearing these mutations exhibit altered splice site and/or exon recognition preferences relative to their wild-type counterparts, resulting in cancer-specific mis-splicing. Such changes in the splicing machinery may create novel vulnerabilities in cancer cells that can be therapeutically exploited using compounds that can influence the splicing process. Further studies to dissect the biochemical, genomic and biological effects of spliceosomal mutations are crucial for the development of cancer therapies targeted at these mutations. PMID:27282250

  8. Female-specific insect lethality engineered using alternative splicing.

    PubMed

    Fu, Guoliang; Condon, Kirsty C; Epton, Matthew J; Gong, Peng; Jin, Li; Condon, George C; Morrison, Neil I; Dafa'alla, Tarig H; Alphey, Luke

    2007-03-01

    The Sterile Insect Technique is a species-specific and environmentally friendly method of pest control involving mass release of sterilized insects that reduce the wild population through infertile matings. Insects carrying a female-specific autocidal genetic system offer an attractive alternative to conventional sterilization methods while also eliminating females from the release population. We exploited sex-specific alternative splicing in insects to engineer female-specific autocidal genetic systems in the Mediterranean fruit fly, Ceratitis capitata. These rely on the insertion of cassette exons from the C. capitata transformer gene into a heterologous tetracycline-repressible transactivator such that the transactivator transcript is disrupted in male splice variants but not in the female-specific one. As the key components of these systems function across a broad phylogenetic range, this strategy addresses the paucity of sex-specific expression systems (e.g., early-acting, female-specific promoters) in insects other than Drosophila melanogaster. The approach may have wide applicability for regulating gene expression in other organisms, particularly for combinatorial control with appropriate promoters. PMID:17322873

  9. UnSplicer: mapping spliced RNA-Seq reads in compact genomes and filtering noisy splicing.

    PubMed

    Burns, Paul D; Li, Yang; Ma, Jian; Borodovsky, Mark

    2014-02-01

    Accurate mapping of spliced RNA-Seq reads to genomic DNA has been known as a challenging problem. Despite significant efforts invested in developing efficient algorithms, with the human genome as a primary focus, the best solution is still not known. A recently introduced tool, TrueSight, has demonstrated better performance compared with earlier developed algorithms such as TopHat and MapSplice. To improve detection of splice junctions, TrueSight uses information on statistical patterns of nucleotide ordering in intronic and exonic DNA. This line of research led to yet another new algorithm, UnSplicer, designed for eukaryotic species with compact genomes where functional alternative splicing is likely to be dominated by splicing noise. Genome-specific parameters of the new algorithm are generated by GeneMark-ES, an ab initio gene prediction algorithm based on unsupervised training. UnSplicer shares several components with TrueSight; the difference lies in the training strategy and the classification algorithm. We tested UnSplicer on RNA-Seq data sets of Arabidopsis thaliana, Caenorhabditis elegans, Cryptococcus neoformans and Drosophila melanogaster. We have shown that splice junctions inferred by UnSplicer are in better agreement with knowledge accumulated on these well-studied genomes than predictions made by earlier developed tools. PMID:24259430

  10. Multiple splicing pathways of group II trans-splicing introns in wheat mitochondria.

    PubMed

    Massel, Karen; Silke, Jordan R; Bonen, Linda

    2016-05-01

    Trans-splicing of discontinuous introns in plant mitochondria requires the assembly of independently-transcribed precursor RNAs into splicing-competent structures, and they are expected to be excised as Y-branched molecules ("broken lariats") because these introns belong to the group II ribozyme family. We now demonstrate that this is just one of several trans-splicing pathways for wheat mitochondrial nad1 intron 4 and nad5 intron 2; they also use a hydrolytic pathway and the liberated 5'-half-intron linear molecules are unexpectedly abundant in the RNA population. We also observe a third productive splicing pathway for nad5 intron 2 that yields full-length excised introns in which the termini are joined in vivo and possess non-encoded nucleotides. In the case of trans-splicing nad1 intron 1, which has a weakly-structured and poorly-conserved core sequence, excision appears to be solely through a hydrolytic pathway. When wheat embryos are germinated in the cold rather than at room temperature, an increased complexity in trans-splicing products is seen for nad1 intron 4, suggesting that there can be environmental effects on the RNA folding of bipartite introns. Our observations provide insights into intron evolution and the complexity of RNA processing events in plant mitochondria. PMID:26970277

  11. Regulation of alternative splicing of CD45 by antagonistic effects of SR protein splicing factors.

    PubMed

    ten Dam, G B; Zilch, C F; Wallace, D; Wieringa, B; Beverley, P C; Poels, L G; Screaton, G R

    2000-05-15

    CD45 is a transmembrane glycoprotein possessing tyrosine phosphatase activity, which is involved in cell signaling. CD45 is expressed on the surface of most leukocytes and can be alternatively spliced by the inclusion or skipping of three variable exons (4, 5, and 6 or A, B, and C) to produce up to eight isoforms. In T cells, the splicing pattern of CD45 isoforms changes after activation; naive cells express high m.w. isoforms of CD45 which predominantly express exon A (CD45RA), whereas activated cells lose expression of exon A to form low m.w. isoforms of CD45 including CD45RO. Little is known about the specific factors controlling the switch in CD45 splicing which occurs on activation. In this study, we examined the influence of the SR family of splicing factors, which, like CD45, are expressed in tissue-specific patterns and have been shown to modulate the alternative splicing of a variety of transcripts. We show that specific SR proteins have antagonistic effects on CD45 splicing, leading either to exon inclusion or skipping. Furthermore, we were able to demonstrate specific changes in the SR protein expression pattern during T cell activation. PMID:10799890

  12. Splicing factor hnRNPH drives an oncogenic splicing switch in gliomas

    PubMed Central

    LeFave, Clare V; Squatrito, Massimo; Vorlova, Sandra; Rocco, Gina L; Brennan, Cameron W; Holland, Eric C; Pan, Ying-Xian; Cartegni, Luca

    2011-01-01

    In tumours, aberrant splicing generates variants that contribute to multiple aspects of tumour establishment, progression and maintenance. We show that in glioblastoma multiforme (GBM) specimens, death-domain adaptor protein Insuloma-Glucagonoma protein 20 (IG20) is consistently aberrantly spliced to generate an antagonist, anti-apoptotic isoform (MAP-kinase activating death domain protein, MADD), which effectively redirects TNF-α/TRAIL-induced death signalling to promote survival and proliferation instead of triggering apoptosis. Splicing factor hnRNPH, which is upregulated in gliomas, controls this splicing event and similarly mediates switching to a ligand-independent, constitutively active Recepteur d′Origine Nantais (RON) tyrosine kinase receptor variant that promotes migration and invasion. The increased cell death and the reduced invasiveness caused by hnRNPH ablation can be rescued by the targeted downregulation of IG20/MADD exon 16- or RON exon 11-containing variants, respectively, using isoform-specific knockdown or splicing redirection approaches. Thus, hnRNPH activity appears to be involved in the pathogenesis and progression of malignant gliomas as the centre of a splicing oncogenic switch, which might reflect reactivation of stem cell patterns and mediates multiple key aspects of aggressive tumour behaviour, including evasion from apoptosis and invasiveness. PMID:21915099

  13. Single Mode Fiber Optic Connectors And Splices

    NASA Astrophysics Data System (ADS)

    Woods, John G.

    1984-08-01

    There is a trend toward increasing use of single mode transmission, particularly in telecommunications where high data bit rates are transmitted for long distances. Inter-connections of multimode fibers can be made in a number of ways, using ferrules, v-grooves, elastomeric splices, etc. However, the connection of single mode fibers, which have core diameters of 4 to 13 μm, requires more precise alignment than do the multimode fibers having core diameters of 50 μm or more. At TRW, we have adapted the four rod alignment guide concept for single mode fiber inter-connections. The principle of this OPTAGUIDE* alignment guide is presented. The single mode connectors and splices use the four rod scheme with an index matching material to eliminate or reduce the losses incurred through fiber end roughness or angularity. We are able to produce demountable connectors for 80/4.4 pm fibers having typical insertion losses of 1.0dB. The main factors in obtaining this result are the naturally precise fiber alignment provided by the alignment guide, and the ability of several manufacturers to maintain tight diametral and core offset tolerances. The single mode OPTALIGN* SM Connectors have been subjected to performance and environmental tests including repeated matings, temperature cycle and vibration. The results of these tests are described in this paper. A feature of the OPTALIGN* SM Connectors is the relative ease and speed of attachment to fiber optic cable in the field, without the use of epoxy or polishing procedures. The alignment guide concept has also been applied to permanent single mode splices. The splicing procedure is simple to perform in the field without expensive or delicate equipment. Construction and assembly procedures of the demountable connectors and permanent splices will be described with the aid of diagrams and photographs.

  14. Widespread alternative and aberrant splicing revealed by lariat sequencing

    PubMed Central

    Stepankiw, Nicholas; Raghavan, Madhura; Fogarty, Elizabeth A.; Grimson, Andrew; Pleiss, Jeffrey A.

    2015-01-01

    Alternative splicing is an important and ancient feature of eukaryotic gene structure, the existence of which has likely facilitated eukaryotic proteome expansions. Here, we have used intron lariat sequencing to generate a comprehensive profile of splicing events in Schizosaccharomyces pombe, amongst the simplest organisms that possess mammalian-like splice site degeneracy. We reveal an unprecedented level of alternative splicing, including alternative splice site selection for over half of all annotated introns, hundreds of novel exon-skipping events, and thousands of novel introns. Moreover, the frequency of these events is far higher than previous estimates, with alternative splice sites on average activated at ∼3% the rate of canonical sites. Although a subset of alternative sites are conserved in related species, implying functional potential, the majority are not detectably conserved. Interestingly, the rate of aberrant splicing is inversely related to expression level, with lowly expressed genes more prone to erroneous splicing. Although we validate many events with RNAseq, the proportion of alternative splicing discovered with lariat sequencing is far greater, a difference we attribute to preferential decay of aberrantly spliced transcripts. Together, these data suggest the spliceosome possesses far lower fidelity than previously appreciated, highlighting the potential contributions of alternative splicing in generating novel gene structures. PMID:26261211

  15. Staying on Message: Ensuring Fidelity in Pre-mRNA Splicing

    PubMed Central

    Semlow, Daniel R.; Jonathan P. Staley

    2012-01-01

    The faithful expression of genes requires that cellular machinery select substrates with high specificity at each step in gene expression. High specificity is particularly important at the stage of nuclear pre-mRNA splicing, during which the spliceosome selects splice sites and excises intervening introns. With low specificity, the usage of alternative sites would yield insertions, deletions and frame shifts in messenger RNA. Recently, biochemical, genetic and genome-wide approaches have significantly advanced our understanding of splicing fidelity. In particular, we have learned that DExD/H-box ATPases play a general role in rejecting and discarding suboptimal substrates and that these factors serve as a paradigm for proofreading NTPases in other systems. Recent advances have also defined fundamental questions for future investigations. PMID:22564363

  16. Staying on message: ensuring fidelity in pre-mRNA splicing.

    PubMed

    Semlow, Daniel R; Staley, Jonathan P

    2012-07-01

    The faithful expression of genes requires that cellular machinery select substrates with high specificity at each step in gene expression. High specificity is particularly important at the stage of nuclear pre-mRNA splicing, during which the spliceosome selects splice sites and excises intervening introns. With low specificity, the usage of alternative sites would yield insertions, deletions and frame shifts in mRNA. Recently, biochemical, genetic and genome-wide approaches have significantly advanced our understanding of splicing fidelity. In particular, we have learned that DExD/H-box ATPases play a general role in rejecting and discarding suboptimal substrates and that these factors serve as a paradigm for proofreading NTPases in other systems. Recent advances have also defined fundamental questions for future investigations. PMID:22564363

  17. Regulation of axon regeneration by the RNA repair and splicing pathway.

    PubMed

    Song, Yuanquan; Sretavan, David; Salegio, Ernesto A; Berg, Jim; Huang, Xi; Cheng, Tong; Xiong, Xin; Meltzer, Shan; Han, Chun; Nguyen, Trong-Tuong; Bresnahan, Jacqueline C; Beattie, Michael S; Jan, Lily Yeh; Jan, Yuh Nung

    2015-06-01

    Mechanisms governing a neuron's regenerative ability are important but not well understood. We identify Rtca (RNA 3'-terminal phosphate cyclase) as an inhibitor of axon regeneration. Removal of Rtca cell-autonomously enhanced axon regrowth in the Drosophila CNS, whereas its overexpression reduced axon regeneration in the periphery. Rtca along with the RNA ligase Rtcb and its catalyst Archease operate in the RNA repair and splicing pathway important for stress-induced mRNA splicing, including that of Xbp1, a cellular stress sensor. Drosophila Rtca and Archease had opposing effects on Xbp1 splicing, and deficiency of Archease or Xbp1 impeded axon regeneration in Drosophila. Moreover, overexpressing mammalian Rtca in cultured rodent neurons reduced axonal complexity in vitro, whereas reducing its function promoted retinal ganglion cell axon regeneration after optic nerve crush in mice. Our study thus links axon regeneration to cellular stress and RNA metabolism, revealing new potential therapeutic targets for treating nervous system trauma. PMID:25961792

  18. Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges

    PubMed Central

    Lovci, Michael T; Ghanem, Dana; Marr, Henry; Arnold, Justin; Gee, Sherry; Parra, Marilyn; Liang, Tiffany Y; Stark, Thomas J; Gehman, Lauren T; Hoon, Shawn; Massirer, Katlin B; Pratt, Gabriel A; Black, Douglas L; Gray, Joe W; Conboy, John G; Yeo, Gene W

    2014-01-01

    Alternative splicing (AS) enables programmed diversity of gene expression across tissues and development. We show here that binding in distal intronic regions (>500 nucleotides (nt) from any exon) by Rbfox splicing factors important in development is extensive and is an active mode of splicing regulation. Similarly to exon-proximal sites, distal sites contain evolutionarily conserved GCATG sequences and are associated with AS activation and repression upon modulation of Rbfox abundance in human and mouse experimental systems. As a proof of principle, we validated the activity of two specific Rbfox enhancers in KIF21A and ENAH distal introns and showed that a conserved long-range RNA-RNA base-pairing interaction (an RNA bridge) is necessary for Rbfox-mediated exon inclusion in the ENAH gene. Thus we demonstrate a previously unknown RNA-mediated mechanism for AS control by distally bound RNA-binding proteins. PMID:24213538

  19. Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges.

    PubMed

    Lovci, Michael T; Ghanem, Dana; Marr, Henry; Arnold, Justin; Gee, Sherry; Parra, Marilyn; Liang, Tiffany Y; Stark, Thomas J; Gehman, Lauren T; Hoon, Shawn; Massirer, Katlin B; Pratt, Gabriel A; Black, Douglas L; Gray, Joe W; Conboy, John G; Yeo, Gene W

    2013-12-01

    Alternative splicing (AS) enables programmed diversity of gene expression across tissues and development. We show here that binding in distal intronic regions (>500 nucleotides (nt) from any exon) by Rbfox splicing factors important in development is extensive and is an active mode of splicing regulation. Similarly to exon-proximal sites, distal sites contain evolutionarily conserved GCATG sequences and are associated with AS activation and repression upon modulation of Rbfox abundance in human and mouse experimental systems. As a proof of principle, we validated the activity of two specific Rbfox enhancers in KIF21A and ENAH distal introns and showed that a conserved long-range RNA-RNA base-pairing interaction (an RNA bridge) is necessary for Rbfox-mediated exon inclusion in the ENAH gene. Thus we demonstrate a previously unknown RNA-mediated mechanism for AS control by distally bound RNA-binding proteins. PMID:24213538

  20. The splicing regulator Rbfox1 (A2BP1) controls neuronal excitation in the mammalian brain

    PubMed Central

    Gehman, Lauren T.; Stoilov, Peter; Maguire, Jamie; Damianov, Andrey; Lin, Chia-Ho; Shiue, Lily; Ares, Manuel; Mody, Istvan; Black, Douglas L.

    2011-01-01

    The Rbfox family of RNA binding proteins regulates alternative splicing of many important neuronal transcripts but their role in neuronal physiology is not clear1. We show here that central nervous system (CNS)-specific deletion of the Rbfox1 gene results in heightened susceptibility to spontaneous and kainic acid-induced seizures. Electrophysiological recording reveals a corresponding increase in neuronal excitability in the dentate gyrus of the knockout mice. Whole transcriptome analyses identify multiple splicing changes in the Rbfox1−/− brain with few changes in overall transcript abundance. These splicing changes alter proteins that mediate synaptic transmission and membrane excitation, some of which are implicated in human epilepsy. Thus, Rbfox1 directs a genetic program required in the prevention of neuronal hyperexcitation and seizures. The Rbfox1 knockout mice provide a new model to study the post-transcriptional regulation of synaptic function. PMID:21623373

  1. NOVA regulates Dcc alternative splicing during neuronal migration and axon guidance in the spinal cord

    PubMed Central

    Leggere, Janelle C; Saito, Yuhki; Darnell, Robert B; Tessier-Lavigne, Marc; Junge, Harald J; Chen, Zhe

    2016-01-01

    RNA-binding proteins (RBPs) control multiple aspects of post-transcriptional gene regulation and function during various biological processes in the nervous system. To further reveal the functional significance of RBPs during neural development, we carried out an in vivo RNAi screen in the dorsal spinal cord interneurons, including the commissural neurons. We found that the NOVA family of RBPs play a key role in neuronal migration, axon outgrowth, and axon guidance. Interestingly, Nova mutants display similar defects as the knockout of the Dcc transmembrane receptor. We show here that Nova deficiency disrupts the alternative splicing of Dcc, and that restoring Dcc splicing in Nova knockouts is able to rescue the defects. Together, our results demonstrate that the production of DCC splice variants controlled by NOVA has a crucial function during many stages of commissural neuron development. DOI: http://dx.doi.org/10.7554/eLife.14264.001 PMID:27223328

  2. A Conserved Nuclear Cyclophilin Is Required for Both RNA Polymerase II Elongation and Co-transcriptional Splicing in Caenorhabditis elegans

    PubMed Central

    Ahn, Jeong H.; Rechsteiner, Andreas; Strome, Susan; Kelly, William G.

    2016-01-01

    The elongation phase of transcription by RNA Polymerase II (Pol II) involves numerous events that are tightly coordinated, including RNA processing, histone modification, and chromatin remodeling. RNA splicing factors are associated with elongating Pol II, and the interdependent coupling of splicing and elongation has been documented in several systems. Here we identify a conserved, multi-domain cyclophilin family member, SIG-7, as an essential factor for both normal transcription elongation and co-transcriptional splicing. In embryos depleted for SIG-7, RNA levels for over a thousand zygotically expressed genes are substantially reduced, Pol II becomes significantly reduced at the 3’ end of genes, marks of transcription elongation are reduced, and unspliced mRNAs accumulate. Our findings suggest that SIG-7 plays a central role in both Pol II elongation and co-transcriptional splicing and may provide an important link for their coordination and regulation. PMID:27541139

  3. A Conserved Nuclear Cyclophilin Is Required for Both RNA Polymerase II Elongation and Co-transcriptional Splicing in Caenorhabditis elegans.

    PubMed

    Ahn, Jeong H; Rechsteiner, Andreas; Strome, Susan; Kelly, William G

    2016-08-01

    The elongation phase of transcription by RNA Polymerase II (Pol II) involves numerous events that are tightly coordinated, including RNA processing, histone modification, and chromatin remodeling. RNA splicing factors are associated with elongating Pol II, and the interdependent coupling of splicing and elongation has been documented in several systems. Here we identify a conserved, multi-domain cyclophilin family member, SIG-7, as an essential factor for both normal transcription elongation and co-transcriptional splicing. In embryos depleted for SIG-7, RNA levels for over a thousand zygotically expressed genes are substantially reduced, Pol II becomes significantly reduced at the 3' end of genes, marks of transcription elongation are reduced, and unspliced mRNAs accumulate. Our findings suggest that SIG-7 plays a central role in both Pol II elongation and co-transcriptional splicing and may provide an important link for their coordination and regulation. PMID:27541139

  4. AGE AND GENDER-DEPENDENT ALTERNATIVE SPLICING OF P/Q-TYPE CALCIUM CHANNEL EF-HAND

    PubMed Central

    Chang, Siao Yun; Yong, Tan Fong; Yu, Chye Yun; Liang, Mui Cheng; Pletnikova, Olga; Troncoso, Juan C.; Burgunder, Jean-Marc; Soong, Tuck Wah

    2007-01-01

    Cav2.1 Ca2+ channels (P/Q-type), which participate in various key roles in the central nervous systems by mediating calcium influx, are extensively spliced. One of its alternatively-spliced exon is 37, which forms part of the EF hand. The expression of exon 37a (EFa form), but not exon 37b (EFb form), confers the channel an activity-dependent enhancement of channel opening known as Ca2+-dependent facilitation (CDF). In this study, we analyzed the trend of EF hand splice variant distributions in mouse, rat and human brain tissues. We observed a developmental switch in rodents, as well as an age and gender bias in human brain tissues, suggestive of a possible role of these EF hand splice variants in neurophysiological specialization. A parallel study performed on rodent brains showed that the data drawn from human and rodent tissues may not necessarily correlate in the process of aging. PMID:17291689

  5. Cauliflower mosaic virus Transcriptome Reveals a Complex Alternative Splicing Pattern

    PubMed Central

    Bouton, Clément; Geldreich, Angèle; Ramel, Laëtitia; Ryabova, Lyubov A.; Dimitrova, Maria; Keller, Mario

    2015-01-01

    The plant pararetrovirus Cauliflower mosaic virus (CaMV) uses alternative splic-ing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5’ region and sug-gested that the main role of CaMV splicing is to downregulate expression of open read-ing frames (ORFs) I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern, as we identify new splice donor and acceptor sites whose selection leads to more than thirteen 35S RNA isoforms in infected turnip plants. Inactivating splice donor or acceptor sites is not lethal for the virus, since disrupted sites are systematically rescued by the activation of cryptic and/or seldom used splice sites. Taken together, our data depict a conserved, complex and flexible process, involving multiple sites, that ensures splicing of 35S RNA. PMID:26162084

  6. IRAS: High-Throughput Identification of Novel Alternative Splicing Regulators.

    PubMed

    Zheng, S

    2016-01-01

    Alternative splicing is a fundamental regulatory process of gene expression. Defects in alternative splicing can lead to various diseases, and modification of disease-causing splicing events presents great therapeutic promise. Splicing outcome is commonly affected by extracellular stimuli and signaling cascades that converge on RNA-binding splicing regulators. These trans-acting factors recognize cis-elements in pre-mRNA transcripts to affect spliceosome assembly and splice site choices. Identification of these splicing regulators and/or upstream modulators has been difficult and traditionally done by piecemeal. High-throughput screening strategies to find multiple regulators of exon splicing have great potential to accelerate the discovery process, but typically confront low sensitivity and low specificity of screening assays. Here we describe a unique screening strategy, IRAS (identifying regulators of alternative splicing), using a pair of dual-output minigene reporters to allow for sensitive detection of exon splicing changes. Each dual-output reporter produces green fluorescent protein (GFP) and red fluorescent protein (RFP) fluorescent signals to assay the two spliced isoforms exclusively. The two complementary minigene reporters alter GFP/RFP output ratios in the opposite direction in response to splicing change. Applying IRAS in cell-based high-throughput screens allows sensitive and specific identification of splicing regulators and modulators for any alternative exons of interest. In comparison to previous high-throughput screening methods, IRAS substantially enhances the specificity of the screening assay. This strategy significantly eliminates false positives without sacrificing sensitive identification of true regulators of splicing. PMID:27241759

  7. Genomic features defining exonic variants that modulate splicing

    PubMed Central

    2010-01-01

    Background Single point mutations at both synonymous and non-synonymous positions within exons can have severe effects on gene function through disruption of splicing. Predicting these mutations in silico purely from the genomic sequence is difficult due to an incomplete understanding of the multiple factors that may be responsible. In addition, little is known about which computational prediction approaches, such as those involving exonic splicing enhancers and exonic splicing silencers, are most informative. Results We assessed the features of single-nucleotide genomic variants verified to cause exon skipping and compared them to a large set of coding SNPs common in the human population, which are likely to have no effect on splicing. Our findings implicate a number of features important for their ability to discriminate splice-affecting variants, including the naturally occurring density of exonic splicing enhancers and exonic splicing silencers of the exon and intronic environment, extensive changes in the number of predicted exonic splicing enhancers and exonic splicing silencers, proximity to the splice junctions and evolutionary constraint of the region surrounding the variant. By extending this approach to additional datasets, we also identified relevant features of variants that cause increased exon inclusion and ectopic splice site activation. Conclusions We identified a number of features that have statistically significant representation among exonic variants that modulate splicing. These analyses highlight putative mechanisms responsible for splicing outcome and emphasize the role of features important for exon definition. We developed a web-tool, Skippy, to score coding variants for these relevant splice-modulating features. PMID:20158892

  8. Dystrophin expression in muscle following gene transfer with a fully deleted ("gutted") adenovirus is markedly improved by trans-acting adenoviral gene products.

    PubMed

    Gilbert, R; Nalbantoglu, J; Howell, J M; Davies, L; Fletcher, S; Amalfitano, A; Petrof, B J; Kamen, A; Massie, B; Karpati, G

    2001-09-20

    Helper-dependent adenoviruses (HDAd) are Ad vectors lacking all or most viral genes. They hold great promise for gene therapy of diseases such as Duchenne muscular dystrophy (DMD), because they are less immunogenic than E1/E3-deleted Ad (first-generation Ad or FGAd) and can carry the full-length (Fl) dystrophin (dys) cDNA (12 kb). We have compared the transgene expression of a HDAd (HDAdCMVDysFl) and a FGAd (FGAdCMV-dys) in cell culture (HeLa, C2C12 myotubes) and in the muscle of mdx mice (the mouse model for DMD). Both vectors encoded dystrophin regulated by the same cytomegalovirus (CMV) promoter. We demonstrate that the amount of dystrophin expressed was significantly higher after gene transfer with FGAdCMV-dys compared to HDAdCMVDysFl both in vitro and in vivo. However, gene transfer with HDAdCMVDysFl in the presence of a FGAd resulted in a significant increase of dystrophin expression indicating that gene products synthesized by the FGAd increase, in trans, the amount of dystrophin produced. This enhancement occurred in cell culture and after gene transfer in the muscle of mdx mice and dystrophic golden retriever (GRMD) dogs, another animal model for DMD. The E4 region of Ad is required for the enhancement, because no increase of dystrophin expression from HDAdCMVDysFl was observed in the presence of an E1/E4-deleted Ad in vitro and in vivo. The characterization of these enhancing gene products followed by their inclusion into an HDAd may be required to produce sufficient dystrophin to mitigate the pathology of DMD by HDAd-mediated gene transfer. PMID:11560768

  9. A serine–arginine-rich (SR) splicing factor modulates alternative splicing of over a thousand genes in Toxoplasma gondii

    PubMed Central

    Yeoh, Lee M.; Goodman, Christopher D.; Hall, Nathan E.; van Dooren, Giel G.; McFadden, Geoffrey I.; Ralph, Stuart A.

    2015-01-01

    Single genes are often subject to alternative splicing, which generates alternative mature mRNAs. This phenomenon is widespread in animals, and observed in over 90% of human genes. Recent data suggest it may also be common in Apicomplexa. These parasites have small genomes, and economy of DNA is evolutionarily favoured in this phylum. We investigated the mechanism of alternative splicing in Toxoplasma gondii, and have identified and localized TgSR3, a homologue of ASF/SF2 (alternative-splicing factor/splicing factor 2, a serine-arginine–rich, or SR protein) to a subnuclear compartment. In addition, we conditionally overexpressed this protein, which was deleterious to growth. qRT-PCR was used to confirm perturbation of splicing in a known alternatively-spliced gene. We performed high-throughput RNA-seq to determine the extent of splicing modulated by this protein. Current RNA-seq algorithms are poorly suited to compact parasite genomes, and hence we complemented existing tools by writing a new program, GeneGuillotine, that addresses this deficiency by segregating overlapping reads into distinct genes. In order to identify the extent of alternative splicing, we released another program, JunctionJuror, that detects changes in intron junctions. Using this program, we identified about 2000 genes that were constitutively alternatively spliced in T. gondii. Overexpressing the splice regulator TgSR3 perturbed alternative splicing in over 1000 genes. PMID:25870410

  10. A serine-arginine-rich (SR) splicing factor modulates alternative splicing of over a thousand genes in Toxoplasma gondii.

    PubMed

    Yeoh, Lee M; Goodman, Christopher D; Hall, Nathan E; van Dooren, Giel G; McFadden, Geoffrey I; Ralph, Stuart A

    2015-05-19

    Single genes are often subject to alternative splicing, which generates alternative mature mRNAs. This phenomenon is widespread in animals, and observed in over 90% of human genes. Recent data suggest it may also be common in Apicomplexa. These parasites have small genomes, and economy of DNA is evolutionarily favoured in this phylum. We investigated the mechanism of alternative splicing in Toxoplasma gondii, and have identified and localized TgSR3, a homologue of ASF/SF2 (alternative-splicing factor/splicing factor 2, a serine-arginine-rich, or SR protein) to a subnuclear compartment. In addition, we conditionally overexpressed this protein, which was deleterious to growth. qRT-PCR was used to confirm perturbation of splicing in a known alternatively-spliced gene. We performed high-throughput RNA-seq to determine the extent of splicing modulated by this protein. Current RNA-seq algorithms are poorly suited to compact parasite genomes, and hence we complemented existing tools by writing a new program, GeneGuillotine, that addresses this deficiency by segregating overlapping reads into distinct genes. In order to identify the extent of alternative splicing, we released another program, JunctionJuror, that detects changes in intron junctions. Using this program, we identified about 2000 genes that were constitutively alternatively spliced in T. gondii. Overexpressing the splice regulator TgSR3 perturbed alternative splicing in over 1000 genes. PMID:25870410

  11. Massive Idiosyncratic Exon Skipping Corrects the Nonsense Mutation in Dystrophic Mouse Muscle and Produces Functional Revertant Fibers by Clonal Expansion

    PubMed Central

    Lu, Q.L.; Morris, G.E.; Wilton, S.D.; Ly, T.; Artem'yeva, O.V.; Strong, P.; Partridge, T.A.

    2000-01-01

    Conventionally, nonsense mutations within a gene preclude synthesis of a full-length functional protein. Obviation of such a blockage is seen in the mdx mouse, where despite a nonsense mutation in exon 23 of the dystrophin gene, occasional so-called revertant muscle fibers are seen to contain near-normal levels of its protein product. Here, we show that reversion of dystrophin expression in mdx mice muscle involves unprecedented massive loss of up to 30 exons. We detected several alternatively processed transcripts that could account for some of the revertant dystrophins and could not detect genomic deletion from the region commonly skipped in revertant dystrophin. This, together with exon skipping in two noncontiguous regions, favors aberrant splicing as the mechanism for the restoration of dystrophin, but is hard to reconcile with the clonal idiosyncrasy of revertant dystrophins. Revertant dystrophins retain functional domains and mediate plasmalemmal assembly of the dystrophin-associated glycoprotein complex. Physiological function of revertant fibers is demonstrated by the clonal growth of revertant clusters with age, suggesting that revertant dystrophin could be used as a guide to the construction of dystrophin expression vectors for individual gene therapy. The dystrophin gene in the mdx mouse provides a favored system for study of exon skipping associated with nonsense mutations. PMID:10704448

  12. Identification of three core regions essential for protein splicing of the yeast Vma1 protozyme. A random mutagenesis study of the entire Vma1-derived endonuclease sequence.

    PubMed

    Kawasaki, M; Nogami, S; Satow, Y; Ohya, Y; Anraku, Y

    1997-06-20

    The translation product of the VMA1 gene of Saccharomyces cerevisiae undergoes protein splicing, in which the intervening region is autocatalytically excised and the franking regions are ligated. The splicing reaction is catalyzed essentially by the in-frame insert, VMA1-derived endonuclease (VDE), which is a site-specific endonuclease to mediate gene homing. Previous mutational analysis of the splicing reaction has been concentrated extensively upon the splice junctions. However, it still remains unknown which amino acid residues are crucial for the splicing reaction within the entire region of VDE and its neighboring elements. In this work, a polymerase chain reaction-based random mutagenesis strategy was used to identify such residues throughout the overall intervening sequence of the VMA1 gene. Splicing-defective mutant proteins were initially screened using a bacterial expression system and then analyzed further in yeast cells. Mutations were mapped at the N- and C-terminal splice junctions and around the N-terminal one-third of VDE. We identified four potent mutants that yielded aberrant products with molecular masses of 200, 90, and 80 kDa. We suggest that the conserved His362, newly identified as the essential residue for the splicing reaction, contributes to the first cleavage at the N-terminal junction, whereas His736 assists the second cleavage by Asn cyclization at the C-terminal junction. Mutations in these regions did not appear to destroy the endonuclease activity of VDE. PMID:9188457

  13. Introns, alternative splicing, spliced leader trans-splicing and differential expression of pcna and cyclin in Perkinsus marinus.

    PubMed

    Zhang, Huan; Dungan, Christopher F; Lin, Senjie

    2011-01-01

    To gain understanding on the structure and regulation of growth-related genes of the parasitic alveolatePerkinsus marinus, we analyzed genes encoding proliferating cell nuclear antigen (pcna) and cyclins (cyclin). Comparison of the full-length cDNAs with the corresponding genomic sequences revealedtrans-splicing of the mRNAs of these genes with a conserved 21-22 nt spliced leader. Over 10 copies ofpcnawere detected, with identical gene structures and similar nucleotide (nt) sequences (88-99%), encoding largely identical amino acid sequences (aa). Two distinct types ofcyclin(Pmacyclin1 andPmacyclin2) were identified, with 66-69% nt and 81-85% aa similarities.Pmacyclin2 was organized in tandem repeats, and was alternatively spliced, giving rise to five subtypes of transcripts. For bothpcnaandcyclingenes, 6-10 introns were found. Quantitative RT-PCR assays showed thatpcnaandPmacyclin2 expression levels were low with small variations during a 28-h time course, whereasPmacyclin1 transcript abundance was 10-100 times higher, and increased markedly during active cell division, suggesting that it is a mitoticcyclinand can be a useful growth marker for this species. The gene structure and expression features along with phylogenetic results position this organism between dinoflagellates and apicomplexans, but its definitive affiliation among alveolates requires further studies. PMID:20650682

  14. Evolution of Nova-Dependent Splicing Regulation in the Brain

    PubMed Central

    Živin, Marko; Darnell, Robert B

    2007-01-01

    A large number of alternative exons are spliced with tissue-specific patterns, but little is known about how such patterns have evolved. Here, we study the conservation of the neuron-specific splicing factors Nova1 and Nova2 and of the alternatively spliced exons they regulate in mouse brain. Whereas Nova RNA binding domains are 94% identical across vertebrate species, Nova-dependent splicing silencer and enhancer elements (YCAY clusters) show much greater divergence, as less than 50% of mouse YCAY clusters are conserved at orthologous positions in the zebrafish genome. To study the relation between the evolution of tissue-specific splicing and YCAY clusters, we compared the brain-specific splicing of Nova-regulated exons in zebrafish, chicken, and mouse. The presence of YCAY clusters in lower vertebrates invariably predicted conservation of brain-specific splicing across species, whereas their absence in lower vertebrates correlated with a loss of alternative splicing. We hypothesize that evolution of Nova-regulated splicing in higher vertebrates proceeds mainly through changes in cis-acting elements, that tissue-specific splicing might in some cases evolve in a single step corresponding to evolution of a YCAY cluster, and that the conservation level of YCAY clusters relates to the functions encoded by the regulated RNAs. PMID:17937501

  15. Alternative Splicing Variants and DNA Methylation Status of BDNF in Inbred Chicken Lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Brain derived neurotrophic factor (BDNF) plays essential roles in neuronal survival and differentiation, synaptic plasticity, central regulation of energy homeostasis, and neuronal development of the central and peripheral nerve system. Here, we report two new splicing variants of the chicken BDNF g...

  16. ALTERNATE PATCHED SPLICE FORMS ARE EXPRESSED IN A TISSUE SPECIFIC MANNER DURING EARLY EMBRYONIC DEVELOPMENT

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND: The Hedgehog (Hh) pathway is critical for embryonic patterning of nearly every organ system in the developing fetus and is highly conserved across phylogeny. We have previously characterized three alternate splice forms of the Ptc gene, including a novel Exon 1C isoform in the mouse, but...

  17. Entropic contributions to the splicing process

    NASA Astrophysics Data System (ADS)

    Osella, Matteo; Caselle, Michele

    2009-12-01

    It has been recently argued that depletion attraction may play an important role in different aspects of cellular organization, ranging from the organization of transcriptional activity in transcription factories to the formation of nuclear bodies. In this paper, we suggest a new application of these ideas in the context of the splicing process, a crucial step of messenger RNA maturation in eukaryotes. We shall show that entropy effects and the resulting depletion attraction may explain the relevance of the aspecific intron length variable in the choice of splice-site recognition modality. On top of that, some qualitative features of the genome architecture of higher eukaryotes can find evolutionary realistic motivation in the light of our model.

  18. Intravitreal Injection of Splice-switching Oligonucleotides to Manipulate Splicing in Retinal Cells

    PubMed Central

    Gérard, Xavier; Perrault, Isabelle; Munnich, Arnold; Kaplan, Josseline; Rozet, Jean-Michel

    2015-01-01

    Leber congenital amaurosis is a severe hereditary retinal dystrophy responsible for neonatal blindness. The most common disease-causing mutation (c.2991+1655A>G; 10–15%) creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. Recently, we reported that splice-switching oligonucleotides (SSO) allow skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients, supporting the feasibility of a SSO-mediated exon skipping strategy to correct the aberrant splicing. Here, we present data in the wild-type mouse, which demonstrate that intravitreal administration of 2'-OMePS-SSO allows selective alteration of Cep290 splicing in retinal cells, including photoreceptors as shown by successful alteration of Abca4 splicing using the same approach. We show that both SSOs and Cep290 skipped mRNA were detectable for at least 1 month and that intravitreal administration of oligonucleotides did not provoke any serious adverse event. These data suggest that intravitreal injections of SSO should be considered to bypass protein truncation resulting from the c.2991+1655A>G mutation as well as other truncating mutations in genes which like CEP290 or ABCA4 have a mRNA size that exceed cargo capacities of US Food and Drug Administration (FDA)-approved adeno-associated virus (AAV)-vectors, thus hampering gene augmentation therapy. PMID:26325627

  19. Exonic Splicing Mutations Are More Prevalent than Currently Estimated and Can Be Predicted by Using In Silico Tools

    PubMed Central

    Soukarieh, Omar; Gaildrat, Pascaline; Hamieh, Mohamad; Drouet, Aurélie; Baert-Desurmont, Stéphanie; Frébourg, Thierry; Tosi, Mario; Martins, Alexandra

    2016-01-01

    The identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient’s RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants), including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs). We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZEI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases. PMID:26761715

  20. A Novel Pathway for Sensory-Mediated Arousal Involves Splicing of an Intron in the period Clock Gene

    PubMed Central

    Cao, Weihuan; Edery, Isaac

    2015-01-01

    Study Objectives: D. melanogaster is an excellent animal model to study how the circadian (≅ 24-h) timing system and sleep regulate daily wake-sleep cycles. Splicing of a temperature-sensitive 3'-terminal intron (termed dmpi8) from the circadian clock gene period (per) regulates the distribution of daily activity in Drosophila. The role of dmpi8 splicing on daily behavior was further evaluated by analyzing sleep. Design: Transgenic flies of the same genetic background but expressing either a wild-type recombinant per gene or one where the efficiency of dmpi8 splicing was increased were exposed to different temperatures in daily light-dark cycles and sleep parameters measured. In addition, transgenic flies were briefly exposed to a variety of sensory-mediated stimuli to measure arousal responses. Results: Surprisingly, we show that the effect of dmpi8 splicing on daytime activity levels does not involve a circadian role for per but is linked to adjustments in sensory-dependent arousal and sleep behavior. Genetically altered flies with high dmpi8 splicing efficiency remain aroused longer following short treatments with light and non-photic cues such as mechanical stimulation. Conclusions: We propose that the thermal regulation of dmpi8 splicing acts as a temperature-calibrated rheostat in a novel arousal mechanism, so that on warm days the inefficient splicing of the dmpi8 intron triggers an increase in quiescence by decreasing sensory-mediated arousal, thus ensuring flies minimize being active during the hot midday sun despite the presence of light in the environment, which is usually a strong arousal cue for diurnal animals. Citation: Cao W, Edery I. A novel pathway for sensory-mediated arousal involves splicing of an intron in the period clock gene. SLEEP 2015;38(1):41–51. PMID:25325457

  1. Lack of dystrophin protein Dp71 results in progressive cataract formation due to loss of fiber cell organization

    PubMed Central

    Darche, Marie; Sahel, José-Alain; Rendon, Alvaro; Tadayoni, Ramin

    2014-01-01

    Purpose Dp71 is the main product of the Duchenne muscular dystrophy (DMD) gene in the central nervous system. While studying the impact of its absence on retinal functions, we discovered that mice lacking Dp71 also developed a progressive opacification of the crystalline lens. The purpose of this study was to perform a detailed characterization of the cataract formation in Dp71 knockout (KO-Dp71) mice. Methods Cataract formations in KO-Dp71 mice and wild-type (wt) littermates were assessed in vivo by slit-lamp examination and ex vivo by histological analysis as a function of aging. The expression and cellular localization of the DMD gene products were monitored by western blot and immunohistochemical analysis. Fiber cell integrity was assessed by analyzing the actin cytoskeleton as well as the expression of aquaporin-0 (AQP0). Results As expected, a slit-lamp examination revealed that only one of the 20 tested wt animals presented with a mild opacification of the lens and only at the most advanced age. However, a lack of Dp71 was associated with a 40% incidence of cataracts as early as 2 months of age, which progressively increased to full penetrance by 7 months. A subsequent histological analysis revealed an alteration in the structures of the lenses of KO-Dp71 mice that correlated with the severity of the lens opacity. An analysis of the expression of the different dystrophin gene products revealed that Dp71 was the major DMD gene product expressed in the lens, especially in fiber cells. The role of Dp71 in fiber cells was also suggested by the progressive disorganization of the lens fibers, which was observed in the absence of Dp71 and demonstrated by irregular staining of the actin network and the aqueous channel AQP0. Conclusions While its role in the retina has been well characterized, this study demonstrates for the first time the role played by Dp71 in a different ocular tissue: the crystalline lens. It primarily demonstrates the role that Dp71 plays in the

  2. MLC1 is associated with the Dystrophin-Glycoprotein Complex at astrocytic endfeet

    PubMed Central

    Boor, Ilja; Nagtegaal, Machiel; Kamphorst, Wouter; van der Valk, Paul; Pronk, Jan C.; van Horssen, Jack; Dinopoulos, Argirios; Bove, Kevin E.; Pascual-Castroviejo, Ignacio; Muntoni, Francesco; Estévez, Raúl; Scheper, Gert C.

    2007-01-01

    Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a progressive cerebral white matter disease with onset in childhood, caused by mutations in the MLC1 gene. MLC1 is a protein with unknown function that is mainly expressed in the brain in astrocytic endfeet at the blood–brain and cerebrospinal fluid–brain barriers. It shares its localization at astrocytic endfeet with the dystrophin-associated glycoprotein complex (DGC). The objective of the present study was to investigate the possible association of MLC1 with the DGC. To test this hypothesis, (co)-localization of DGC-proteins and MLC1 was analyzed by immunohistochemical stainings in gliotic brain tissue from a patient with multiple sclerosis, in glioblastoma tissue and in brain tissue from an MLC patient. In control tissue, a direct protein interaction was tested by immunoprecipitation. Results revealed that MLC1 is co-localized with DGC-proteins in gliotic brain tissue. We demonstrated that both MLC1 and aquaporin-4, a member of the DGC, were redistributed in glioblastoma cells. In MLC brain tissue, we showed absence of MLC1 and altered expression of several DGC-proteins. We demonstrated a direct protein interaction between MLC1 and Kir4.1. From these results we conclude that MLC1 is associated with the DGC at astrocytic endfeet. PMID:17628813

  3. Multi-level omics analysis in a murine model of dystrophin loss and therapeutic restoration

    PubMed Central

    Roberts, Thomas C.; Johansson, Henrik J.; McClorey, Graham; Godfrey, Caroline; Blomberg, K. Emelie M.; Coursindel, Thibault; Gait, Michael J.; Smith, C.I. Edvard; Lehtiö, Janne; EL Andaloussi, Samir; Wood, Matthew J.A.

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a classical monogenic disorder, a model disease for genomic studies and a priority candidate for regenerative medicine and gene therapy. Although the genetic cause of DMD is well known, the molecular pathogenesis of disease and the response to therapy are incompletely understood. Here, we describe analyses of protein, mRNA and microRNA expression in the tibialis anterior of the mdx mouse model of DMD. Notably, 3272 proteins were quantifiable and 525 identified as differentially expressed in mdx muscle (P < 0.01). Therapeutic restoration of dystrophin by exon skipping induced widespread shifts in protein and mRNA expression towards wild-type expression levels, whereas the miRNome was largely unaffected. Comparison analyses between datasets showed that protein and mRNA ratios were only weakly correlated (r = 0.405), and identified a multitude of differentially affected cellular pathways, upstream regulators and predicted miRNA–target interactions. This study provides fundamental new insights into gene expression and regulation in dystrophic muscle. PMID:26385637

  4. Multi-level omics analysis in a murine model of dystrophin loss and therapeutic restoration.

    PubMed

    Roberts, Thomas C; Johansson, Henrik J; McClorey, Graham; Godfrey, Caroline; Blomberg, K Emelie M; Coursindel, Thibault; Gait, Michael J; Smith, C I Edvard; Lehtiö, Janne; El Andaloussi, Samir; Wood, Matthew J A

    2015-12-01

    Duchenne muscular dystrophy (DMD) is a classical monogenic disorder, a model disease for genomic studies and a priority candidate for regenerative medicine and gene therapy. Although the genetic cause of DMD is well known, the molecular pathogenesis of disease and the response to therapy are incompletely understood. Here, we describe analyses of protein, mRNA and microRNA expression in the tibialis anterior of the mdx mouse model of DMD. Notably, 3272 proteins were quantifiable and 525 identified as differentially expressed in mdx muscle (P < 0.01). Therapeutic restoration of dystrophin by exon skipping induced widespread shifts in protein and mRNA expression towards wild-type expression levels, whereas the miRNome was largely unaffected. Comparison analyses between datasets showed that protein and mRNA ratios were only weakly correlated (r = 0.405), and identified a multitude of differentially affected cellular pathways, upstream regulators and predicted miRNA-target interactions. This study provides fundamental new insights into gene expression and regulation in dystrophic muscle. PMID:26385637

  5. Defective T-lymphocyte migration to muscles in dystrophin-deficient mice.

    PubMed

    Cascabulho, Cynthia M; Bani Corrêa, Cristiane; Cotta-de-Almeida, Vinícius; Henriques-Pons, Andrea

    2012-08-01

    Duchenne muscular dystrophy (DMD), an X-linked recessive disorder affecting 1 in 3500 males, is caused by mutations in the dystrophin gene. DMD leads to degeneration of skeletal and cardiac muscles and to chronic inflammation. The mdx/mdx mouse has been widely used to study DMD; this model mimics most characteristics of the disease, including low numbers of T cells in damaged muscles. In this study, we aimed to assess migration of T cells to the heart and to identify any alterations in adhesion molecules that could possibly modulate this process. In 6-week-old mdx/mdx mice, blood leukocytes, including T cells, were CD62L(+), but by 12 weeks of age down-modulation was evident, with only approximately 40% of T cells retaining this molecule. Our in vitro and in vivo results point to a P2X7-dependent shedding of CD62L (with high levels in the serum), which in 12-week-old mdx/mdx mice reduces blood T cell competence to adhere to cardiac vessels in vitro and to reach cardiac tissue in vivo, even after Trypanosoma cruzi infection, a known inducer of lymphoid myocarditis. In mdx/mdx mice treated with Brilliant Blue G, a P2X7 blocker, these blood lymphocytes retained CD62L and were capable of migrating to the heart. These results provide new insights into the mechanisms of inflammatory infiltration and immune regulation in DMD. PMID:22733008

  6. Sequence characterisation of deletion breakpoints in the dystrophin gene by PCR

    SciTech Connect

    Abbs, S.; Sandhu, S.; Bobrow, M.

    1994-09-01

    Partial deletions of the dystrophin gene account for 65% of cases of Duchenne muscular dystrophy. A high proportion of these structural changes are generated by new mutational events, and lie predominantly within two `hotspot` regions, yet the underlying reasons for this are not known. We are characterizing and sequencing the regions surrounding deletion breakpoints in order to: (i) investigate the mechanisms of deletion mutation, and (ii) enable the design of PCR assays to specifically amplify mutant and normal sequences, allowing us to search for the presence of somatic mosaicism in appropriate family members. Using this approach we have been able to demonstrate the presence of somatic mosaicism in a maternal grandfather of a DMD-affected male, deleted for exons 49-50. Three deletions, namely of exons 48-49, 49-50, and 50, have been characterized using a PCR approach that avoids any cloning procedures. Breakpoints were initially localized to within regions of a few kilobases using Southern blot restriction analyses with exon-specific probes and PCR amplification of exonic and intronic loci. Sequencing was performed directly on PCR products: (i) mutant sequences were obtained from long-range or inverse-PCR across the deletion junction fragments, and (ii) normal sequences were obtained from the products of standard PCR, vectorette PCR, or inverse-PCR performed on YACs. Further characterization of intronic sequences will allow us to amplify and sequence across other deletion breakpoints and increase our knowledge of the mechanisms of mutation in the dystophin gene.

  7. Recombinase-mediated reprogramming and dystrophin gene addition in mdx mouse induced pluripotent stem cells.

    PubMed

    Zhao, Chunli; Farruggio, Alfonso P; Bjornson, Christopher R R; Chavez, Christopher L; Geisinger, Jonathan M; Neal, Tawny L; Karow, Marisa; Calos, Michele P

    2014-01-01

    A cell therapy strategy utilizing genetically-corrected induced pluripotent stem cells (iPSC) may be an attractive approach for genetic disorders such as muscular dystrophies. Methods for genetic engineering of iPSC that emphasize precision and minimize random integration would be beneficial. We demonstrate here an approach in the mdx mouse model of Duchenne muscular dystrophy that focuses on the use of site-specific recombinases to achieve genetic engineering. We employed non-viral, plasmid-mediated methods to reprogram mdx fibroblasts, using phiC31 integrase to insert a single copy of the reprogramming genes at a safe location in the genome. We next used Bxb1 integrase to add the therapeutic full-length dystrophin cDNA to the iPSC in a site-specific manner. Unwanted DNA sequences, including the reprogramming genes, were then precisely deleted with Cre resolvase. Pluripotency of the iPSC was analyzed before and after gene addition, and ability of the genetically corrected iPSC to differentiate into myogenic precursors was evaluated by morphology, immunohistochemistry, qRT-PCR, FACS analysis, and intramuscular engraftment. These data demonstrate a non-viral, reprogramming-plus-gene addition genetic engineering strategy utilizing site-specific recombinases that can be applied easily to mouse cells. This work introduces a significant level of precision in the genetic engineering of iPSC that can be built upon in future studies. PMID:24781921

  8. Haplotypes in the Dystrophin DNA Segment Point to a Mosaic Origin of Modern Human Diversity

    PubMed Central

    Ziętkiewicz, Ewa; Yotova, Vania; Gehl, Dominik; Wambach, Tina; Arrieta, Isabel; Batzer, Mark; Cole, David E. C.; Hechtman, Peter; Kaplan, Feige; Modiano, David; Moisan, Jean-Paul; Michalski, Roman; Labuda, Damian

    2003-01-01

    Although Africa has played a central role in human evolutionary history, certain studies have suggested that not all contemporary human genetic diversity is of recent African origin. We investigated 35 simple polymorphic sites and one Tn microsatellite in an 8-kb segment of the dystrophin gene. We found 86 haplotypes in 1,343 chromosomes from around the world. Although a classical out-of-Africa topology was observed in trees based on the variant frequencies, the tree of haplotype sequences reveals three lineages accounting for present-day diversity. The proportion of new recombinants and the diversity of the Tn microsatellite were used to estimate the age of haplotype lineages and the time of colonization events. The lineage that underwent the great expansion originated in Africa prior to the Upper Paleolithic (27,000–56,000 years ago). A second group, of structurally distinct haplotypes that occupy a central position on the tree, has never left Africa. The third lineage is represented by the haplotype that lies closest to the root, is virtually absent in Africa, and appears older than the recent out-of-Africa expansion. We propose that this lineage could have left Africa before the expansion (as early as 160,000 years ago) and admixed, outside of Africa, with the expanding lineage. Contemporary human diversity, although dominated by the recently expanded African lineage, thus represents a mosaic of different contributions. PMID:14513410

  9. Somatodendritic and excitatory postsynaptic distribution of neuron-type dystrophin isoform, Dp40, in hippocampal neurons.

    PubMed

    Fujimoto, Takahiro; Itoh, Kyoko; Yaoi, Takeshi; Fushiki, Shinji

    2014-09-12

    The Duchenne muscular dystrophy (DMD) gene produces multiple dystrophin (Dp) products due to the presence of several promoters. We previously reported the existence of a novel short isoform of Dp, Dp40, in adult mouse brain. However, the exact biochemical expression profile and cytological distribution of the Dp40 protein remain unknown. In this study, we generated a polyclonal antibody against the NH2-terminal region of the Dp40 and identified the expression profile of Dp40 in the mouse brain. Through an analysis using embryonic and postnatal mouse cerebrums, we found that Dp40 emerged from the early neonatal stages until adulthood, whereas Dp71, an another Dp short isoform, was highly detected in both prenatal and postnatal cerebrums. Intriguingly, relative expressions of Dp40 and Dp71 were prominent in cultured dissociated neurons and non-neuronal cells derived from mouse hippocampus, respectively. Furthermore, the immunocytological distribution of Dp40 was analyzed in dissociated cultured neurons, revealing that Dp40 is detected in the soma and its dendrites, but not in the axon. It is worthy to note that Dp40 is localized along the subplasmalemmal region of the dendritic shafts, as well as at excitatory postsynaptic sites. Thus, Dp40 was identified as a neuron-type Dp possibly involving dendritic and synaptic functions. PMID:25152393

  10. SKIP Is a Component of the Spliceosome Linking Alternative Splicing and the Circadian Clock in Arabidopsis[W

    PubMed Central

    Wang, Xiaoxue; Wu, Fangming; Xie, Qiguang; Wang, Huamei; Wang, Ying; Yue, Yanling; Gahura, Ondrej; Ma, Shuangshuang; Liu, Lei; Cao, Ying; Jiao, Yuling; Puta, Frantisek; McClung, C. Robertson; Xu, Xiaodong; Ma, Ligeng

    2012-01-01

    Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for clock function. However, little is known about the molecular mechanisms linking RNA metabolism to the circadian clock network. Here, we report that a conserved SNW/Ski-interacting protein (SKIP) domain protein, SKIP, a splicing factor and component of the spliceosome, is involved in posttranscriptional regulation of circadian clock genes in Arabidopsis thaliana. Mutation in SKIP lengthens the circadian period in a temperature-sensitive manner and affects light input and the sensitivity of the clock to light resetting. SKIP physically interacts with the spliceosomal splicing factor Ser/Arg-rich protein45 and associates with the pre-mRNA of clock genes, such as PSEUDORESPONSE REGULATOR7 (PRR7) and PRR9, and is necessary for the regulation of their alternative splicing and mRNA maturation. Genome-wide investigations reveal that SKIP functions in regulating alternative splicing of many genes, presumably through modulating recognition or cleavage of 5′ and 3′ splice donor and acceptor sites. Our study addresses a fundamental question on how the mRNA splicing machinery contributes to circadian clock function at a posttranscriptional level. PMID:22942380

  11. Novel splice site mutation in the growth hormone receptor gene in Turkish patients with Laron-type dwarfism.

    PubMed

    Arman, Ahmet; Ozon, Alev; Isguven, Pinar S; Coker, Ajda; Peker, Ismail; Yordam, Nursen

    2008-01-01

    Growth hormone (GH) is involved in growth, and fat and carbohydrate metabolism. Interaction of GH with the GH receptor (GHR) is necessary for systemic and local production of insulin-like growth factor-I (IGF-I) which mediates GH actions. Mutations in the GHR cause severe postnatal growth failure; the disorder is an autosomal recessive genetic disease resulting in GH insensitivity, called Laron syndrome. It is characterized by dwarfism with elevated serum GH and low levels of IGF-I. We analyzed the GHR gene for mutations and polymorphisms in eight patients with Laron-type dwarfism from six families. We found three missense mutations (S40L, V125A, I526L), one nonsense mutation (W157X), and one splice site mutation in the extracellular domain of GHR. Furthermore, G168G and exon 3 deletion polymorphisms were detected in patients with Laron syndrome. The splice site mutation, which is a novel mutation, was located at the donor splice site of exon 2/ intron 2 within GHR. Although this mutation changed the highly conserved donor splice site consensus sequence GT to GGT by insertion of a G residue, the intron splicing between exon 2 and exon 3 was detected in the patient. These results imply that the splicing occurs arthe GT site in intron 2, leaving the extra inserted G residue at the end of exon 2, thus changing the open reading frame of GHR resulting in a premature termination codon in exon 3. PMID:18404972

  12. Perturbation of myelin basic protein (Mbp) splice variant expression in developing rat cerebellum following perinatal exposure to methylmercury.

    PubMed

    Padhi, Bhaja K; Pelletier, Guillaume

    2012-09-18

    Myelin sheaths surrounding axons are essential for saltatory conduction of nerve impulse in the central nervous system. A major protein constituent of myelin sheaths is produced by the myelin basic protein (Mbp) gene, whose expression in oligodendrocytes is conserved across vertebrates. In rat, five Mbp splice variants resulting from alternative splicing of exons 2, 5 and/or 6 are characterized. We developed a PCR-based strategy to quantify individual Mbp splice variants and characterized a sixth Mbp splice variant lacking only exon 5. This newly identified splice variant is predominantly expressed in developing rat brain and has orthologs in mouse and human. Many neurotoxic chemicals can perturb myelination and Mbp gene expression. Regulation of Mbp gene expression at the post-transcriptional level was assessed following perinatal exposure to neurotoxic methylmercury (2 mg/kg b.w./day). Similar reductions in total and individual Mbp splice variant mRNA levels suggest that methylmercury-induced perturbation in Mbp gene expression occurred as a consequence of decreased oligodendrocyte cell population in absence of a significant impact on its post-transcriptional regulation. PMID:22835759

  13. Splicing variants of porcine synphilin-1.

    PubMed

    Larsen, Knud; Madsen, Lone Bruhn; Farajzadeh, Leila; Bendixen, Christian

    2015-09-01

    Parkinson's disease (PD), idiopathic and familial, is characterized by degradation of dopaminergic neurons and the presence of Lewy bodies (LB) in the substantia nigra. LBs contain aggregated proteins of which α-synuclein is the major component. The protein synphilin-1 interacts and colocalizes with α-synuclein in LBs. The aim of this study was to isolate and characterize porcine synphilin-1 and isoforms hereof with the future perspective to use the pig as a model for Parkinson's disease. The porcine SNCAIP cDNA was cloned by reverse transcriptase PCR. The spatial expression of SNCAIP mRNA was investigated by RNAseq. The presented work reports the molecular cloning and characterization of the porcine (Sus scrofa) synphilin-1 cDNA (SNCAIP) and three splice variants hereof. The porcine SNCAIP cDNA codes for a protein (synphilin-1) of 919 amino acids which shows a high similarity to human (90%) and to mouse (84%) synphilin-1. Three shorter transcript variants of the synphilin-1 gene were identified, all lacking one or more exons. SNCAIP transcripts were detected in most examined organs and tissues and the highest expression was found in brain tissues and lung. Conserved splicing variants and a novel splice form of synhilin-1 were found in this study. All synphilin-1 isoforms encoded by the identified transcript variants lack functional domains important for protein degradation. PMID:26101749

  14. Vials: Visualizing Alternative Splicing of Genes

    PubMed Central

    Strobelt, Hendrik; Alsallakh, Bilal; Botros, Joseph; Peterson, Brant; Borowsky, Mark; Pfister, Hanspeter; Lex, Alexander

    2016-01-01

    Alternative splicing is a process by which the same DNA sequence is used to assemble different proteins, called protein isoforms. Alternative splicing works by selectively omitting some of the coding regions (exons) typically associated with a gene. Detection of alternative splicing is difficult and uses a combination of advanced data acquisition methods and statistical inference. Knowledge about the abundance of isoforms is important for understanding both normal processes and diseases and to eventually improve treatment through targeted therapies. The data, however, is complex and current visualizations for isoforms are neither perceptually efficient nor scalable. To remedy this, we developed Vials, a novel visual analysis tool that enables analysts to explore the various datasets that scientists use to make judgments about isoforms: the abundance of reads associated with the coding regions of the gene, evidence for junctions, i.e., edges connecting the coding regions, and predictions of isoform frequencies. Vials is scalable as it allows for the simultaneous analysis of many samples in multiple groups. Our tool thus enables experts to (a) identify patterns of isoform abundance in groups of samples and (b) evaluate the quality of the data. We demonstrate the value of our tool in case studies using publicly available datasets. PMID:26529712

  15. Integrating alternative splicing detection into gene prediction

    PubMed Central

    Foissac, Sylvain; Schiex, Thomas

    2005-01-01

    Background Alternative splicing (AS) is now considered as a major actor in transcriptome/proteome diversity and it cannot be neglected in the annotation process of a new genome. Despite considerable progresses in term of accuracy in computational gene prediction, the ability to reliably predict AS variants when there is local experimental evidence of it remains an open challenge for gene finders. Results We have used a new integrative approach that allows to incorporate AS detection into ab initio gene prediction. This method relies on the analysis of genomically aligned transcript sequences (ESTs and/or cDNAs), and has been implemented in the dynamic programming algorithm of the graph-based gene finder EuGÈNE. Given a genomic sequence and a set of aligned transcripts, this new version identifies the set of transcripts carrying evidence of alternative splicing events, and provides, in addition to the classical optimal gene prediction, alternative optimal predictions (among those which are consistent with the AS events detected). This allows for multiple annotations of a single gene in a way such that each predicted variant is supported by a transcript evidence (but not necessarily with a full-length coverage). Conclusions This automatic combination of experimental data analysis and ab initio gene finding offers an ideal integration of alternatively spliced gene prediction inside a single annotation pipeline. PMID:15705189

  16. Antisense Mediated Splicing Modulation For Inherited Metabolic Diseases: Challenges for Delivery

    PubMed Central

    Pérez, Belen; Vilageliu, Lluisa; Grinberg, Daniel

    2014-01-01

    In the past few years, research in targeted mutation therapies has experienced significant advances, especially in the field of rare diseases. In particular, the efficacy of antisense therapy for suppression of normal, pathogenic, or cryptic splice sites has been demonstrated in cellular and animal models and has already reached the clinical trials phase for Duchenne muscular dystrophy. In different inherited metabolic diseases, splice switching oligonucleotides (SSOs) have been used with success in patients' cells to force pseudoexon skipping or to block cryptic splice sites, in both cases recovering normal transcript and protein and correcting the enzyme deficiency. However, future in vivo studies require individual approaches for delivery depending on the gene defect involved, given the different patterns of tissue and organ expression. Herein we review the state of the art of antisense therapy targeting RNA splicing in metabolic diseases, grouped according to their expression patterns—multisystemic, hepatic, or in central nervous system (CNS)—and summarize the recent progress achieved in the field of in vivo delivery of oligonucleotides to each organ or system. Successful body-wide distribution of SSOs and preferential distribution in the liver after systemic administration have been reported in murine models for different diseases, while for CNS limited data are available, although promising results with intratechal injections have been achieved. PMID:24506780

  17. Antisense mediated splicing modulation for inherited metabolic diseases: challenges for delivery.

    PubMed

    Pérez, Belen; Vilageliu, Lluisa; Grinberg, Daniel; Desviat, Lourdes R

    2014-02-01

    In the past few years, research in targeted mutation therapies has experienced significant advances, especially in the field of rare diseases. In particular, the efficacy of antisense therapy for suppression of normal, pathogenic, or cryptic splice sites has been demonstrated in cellular and animal models and has already reached the clinical trials phase for Duchenne muscular dystrophy. In different inherited metabolic diseases, splice switching oligonucleotides (SSOs) have been used with success in patients' cells to force pseudoexon skipping or to block cryptic splice sites, in both cases recovering normal transcript and protein and correcting the enzyme deficiency. However, future in vivo studies require individual approaches for delivery depending on the gene defect involved, given the different patterns of tissue and organ expression. Herein we review the state of the art of antisense therapy targeting RNA splicing in metabolic diseases, grouped according to their expression patterns-multisystemic, hepatic, or in central nervous system (CNS)-and summarize the recent progress achieved in the field of in vivo delivery of oligonucleotides to each organ or system. Successful body-wide distribution of SSOs and preferential distribution in the liver after systemic administration have been reported in murine models for different diseases, while for CNS limited data are available, although promising results with intratechal injections have been achieved. PMID:24506780

  18. Splicing of many human genes involves sites embedded within introns

    PubMed Central

    Kelly, Steven; Georgomanolis, Theodore; Zirkel, Anne; Diermeier, Sarah; O'Reilly, Dawn; Murphy, Shona; Längst, Gernot; Cook, Peter R.; Papantonis, Argyris

    2015-01-01

    The conventional model for splicing involves excision of each intron in one piece; we demonstrate this inaccurately describes splicing in many human genes. First, after switching on transcription of SAMD4A, a gene with a 134 kb-long first intron, splicing joins the 3′ end of exon 1 to successive points within intron 1 well before the acceptor site at exon 2 is made. Second, genome-wide analysis shows that >60% of active genes yield products generated by such intermediate intron splicing. These products are present at ∼15% the levels of primary transcripts, are encoded by conserved sequences similar to those found at canonical acceptors, and marked by distinctive structural and epigenetic features. Finally, using targeted genome editing, we demonstrate that inhibiting the formation of these splicing intermediates affects efficient exon–exon splicing. These findings greatly expand the functional and regulatory complexity of the human transcriptome. PMID:25897131

  19. A general role for splicing enhancers in exon definition.

    PubMed Central

    Lam, Bianca J; Hertel, Klemens J

    2002-01-01

    Exonic splicing enhancers (ESEs) facilitate exon definition by assisting in the recruitment of splicing factors to the adjacent intron. Here we demonstrate that suboptimal 5' and 3' splice sites are activated independently by ESEs when they are located on different exons. However, when they are situated within a single exon, the same weak 5' and 3' splice sites are activated simultaneously by a single ESE. These findings demonstrate that a single ESE promotes the recognition of both exon/intron junctions within the same step during exon definition. Our results suggest that ESEs recruit a multicomponent complex that minimally contains components of the splicing machinery required for 5' and 3' splice site selection. PMID:12403462

  20. Superconducting cable-in-conduit low resistance splice

    DOEpatents

    Artman, Thomas A.

    2003-06-24

    A low resistance splice connects two cable-in-conduit superconductors to each other. Dividing collars for arranging sub-cable units from each conduit are provided, along with clamping collars for mating each sub-cable wire assembly to form mated assemblies. The mated assemblies ideally can be accomplished by way of splicing collar. The mated assemblies are cooled by way of a flow of coolant, preferably helium. A method for implementing such a splicing is also described.

  1. Alternative Splicing and Transcriptome Profiling of Experimental Autoimmune Encephalomyelitis Using Genome-Wide Exon Arrays

    PubMed Central

    Gillett, Alan; Maratou, Klio; Fewings, Chris; Harris, Robert A.; Jagodic, Maja; Aitman, Tim; Olsson, Tomas

    2009-01-01

    Background Multiple Sclerosis (MS) is a chronic inflammatory disease causing demyelination and nerve loss in the central nervous system. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS that is widely used to investigate complex pathogenic mechanisms. Transcriptional control through isoform selection and mRNA levels determines pathway activation and ultimately susceptibility to disease. Methodology/Principal Findings We have studied the role of alternative splicing and differential expression in lymph node cells from EAE-susceptible Dark Agouti (DA) and EAE-resistant Piebald Virol Glaxo.AV1 (PVG) inbred rat strains using Affymetrix Gene Chip Rat Exon 1.0 ST Arrays. Comparing the two strains, we identified 11 differentially spliced and 206 differentially expressed genes at day 7 post-immunization, as well as 9 differentially spliced and 144 differentially expressed genes upon autoantigen re-stimulation. Functional clustering and pathway analysis implicate genes for glycosylation, lymphocyte activation, potassium channel activity and cellular differentiation in EAE susceptibility. Conclusions/Significance Our results demonstrate that alternative splicing occurs during complex disease and may govern EAE susceptibility. Additionally, transcriptome analysis not only identified previously defined EAE pathways regulating the immune system, but also novel mechanisms. Furthermore, several identified genes overlap known quantitative trait loci, providing novel causative candidate targets governing EAE. PMID:19915720

  2. Evolutionary Insights into RNA trans-Splicing in Vertebrates

    PubMed Central

    Lei, Quan; Li, Cong; Zuo, Zhixiang; Huang, Chunhua; Cheng, Hanhua; Zhou, Rongjia

    2016-01-01

    Pre-RNA splicing is an essential step in generating mature mRNA. RNA trans-splicing combines two separate pre-mRNA molecules to form a chimeric non-co-linear RNA, which may exert a function distinct from its original molecules. Trans-spliced RNAs may encode novel proteins or serve as noncoding or regulatory RNAs. These novel RNAs not only increase the complexity of the proteome but also provide new regulatory mechanisms for gene expression. An increasing amount of evidence indicates that trans-splicing occurs frequently in both physiological and pathological processes. In addition, mRNA reprogramming based on trans-splicing has been successfully applied in RNA-based therapies for human genetic diseases. Nevertheless, clarifying the extent and evolution of trans-splicing in vertebrates and developing detection methods for trans-splicing remain challenging. In this review, we summarize previous research, highlight recent advances in trans-splicing, and discuss possible splicing mechanisms and functions from an evolutionary viewpoint. PMID:26966239

  3. RNA Splicing: Regulation and Dysregulation in the Heart.

    PubMed

    van den Hoogenhof, Maarten M G; Pinto, Yigal M; Creemers, Esther E

    2016-02-01

    RNA splicing represents a post-transcriptional mechanism to generate multiple functional RNAs or proteins from a single transcript. The evolution of RNA splicing is a prime example of the Darwinian function follows form concept. A mutation that leads to a new mRNA (form) that encodes for a new functional protein (function) is likely to be retained, and this way, the genome has gradually evolved to encode for genes with multiple isoforms, thereby creating an enormously diverse transcriptome. Advances in technologies to characterize RNA populations have led to a better understanding of RNA processing in health and disease. In the heart, alternative splicing is increasingly being recognized as an important layer of post-transcriptional gene regulation. Moreover, the recent identification of several cardiac splice factors, such as RNA-binding motif protein 20 and SF3B1, not only provided important insight into the mechanisms underlying alternative splicing but also revealed how these splicing factors impact functional properties of the heart. Here, we review our current knowledge of alternative splicing in the heart, with a particular focus on the major and minor spliceosome, the factors controlling RNA splicing, and the role of alternative splicing in cardiac development and disease. PMID:26846640

  4. Impacts of Alternative Splicing Events on the Differentiation of Adipocytes

    PubMed Central

    Lin, Jung-Chun

    2015-01-01

    Alternative splicing was found to be a common phenomenon after the advent of whole transcriptome analyses or next generation sequencing. Over 90% of human genes were demonstrated to undergo at least one alternative splicing event. Alternative splicing is an effective mechanism to spatiotemporally expand protein diversity, which influences the cell fate and tissue development. The first focus of this review is to highlight recent studies, which demonstrated effects of alternative splicing on the differentiation of adipocytes. Moreover, use of evolving high-throughput approaches, such as transcriptome analyses (RNA sequencing), to profile adipogenic transcriptomes, is also addressed. PMID:26389882

  5. Adenosine to Inosine editing frequency controlled by splicing efficiency.

    PubMed

    Licht, Konstantin; Kapoor, Utkarsh; Mayrhofer, Elisa; Jantsch, Michael F

    2016-07-27

    Alternative splicing and adenosine to inosine (A to I) RNA-editing are major factors leading to co- and post-transcriptional modification of genetic information. Both, A to I editing and splicing occur in the nucleus. As editing sites are frequently defined by exon-intron basepairing, mRNA splicing efficiency should affect editing levels. Moreover, splicing rates affect nuclear retention and will therefore also influence the exposure of pre-mRNAs to the editing-competent nuclear environment. Here, we systematically test the influence of splice rates on RNA-editing using reporter genes but also endogenous substrates. We demonstrate for the first time that the extent of editing is controlled by splicing kinetics when editing is guided by intronic elements. In contrast, editing sites that are exclusively defined by exonic structures are almost unaffected by the splicing efficiency of nearby introns. In addition, we show that editing levels in pre- and mature mRNAs do not match. This phenomenon can in part be explained by the editing state of an RNA influencing its splicing rate but also by the binding of the editing enzyme ADAR that interferes with splicing. PMID:27112566

  6. Splice-switching antisense oligonucleotides as therapeutic drugs.

    PubMed

    Havens, Mallory A; Hastings, Michelle L

    2016-08-19

    Splice-switching oligonucleotides (SSOs) are short, synthetic, antisense, modified nucleic acids that base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA-RNA base-pairing or protein-RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Splicing of pre-mRNA is required for the proper expression of the vast majority of protein-coding genes, and thus, targeting the process offers a means to manipulate protein production from a gene. Splicing modulation is particularly valuable in cases of disease caused by mutations that lead to disruption of normal splicing or when interfering with the normal splicing process of a gene transcript may be therapeutic. SSOs offer an effective and specific way to target and alter splicing in a therapeutic manner. Here, we discuss the different approaches used to target and alter pre-mRNA splicing with SSOs. We detail the modifications to the nucleic acids that make them promising therapeutics and discuss the challenges to creating effective SSO drugs. We highlight the development of SSOs designed to treat Duchenne muscular dystrophy and spinal muscular atrophy, which are currently being tested in clinical trials. PMID:27288447

  7. Caveolin-3 null mice show a loss of caveolae, changes in the microdomain distribution of the dystrophin-glycoprotein complex, and t-tubule abnormalities.

    PubMed

    Galbiati, F; Engelman, J A; Volonte, D; Zhang, X L; Minetti, C; Li, M; Hou, H; Kneitz, B; Edelmann, W; Lisanti, M P

    2001-06-15

    Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cells. Recently, we identified a novel autosomal dominant form of limb-girdle muscular dystrophy (LGMD-1C) in humans that is due to mutations within the coding sequence of the human caveolin-3 gene (3p25). These LGMD-1C mutations lead to an approximately 95% reduction in caveolin-3 protein expression, i.e. a caveolin-3 deficiency. Here, we created a caveolin-3 null (CAV3 -/-) mouse model, using standard homologous recombination techniques, to mimic a caveolin-3 deficiency. We show that these mice lack caveolin-3 protein expression and sarcolemmal caveolae membranes. In addition, analysis of skeletal muscle tissue from these caveolin-3 null mice reveals: (i) mild myopathic changes; (ii) an exclusion of the dystrophin-glycoprotein complex from lipid raft domains; and (iii) abnormalities in the organization of the T-tubule system, with dilated and longitudinally oriented T-tubules. These results have clear mechanistic implications for understanding the pathogenesis of LGMD-1C at a molecular level. PMID:11259414

  8. Screening Duchenne and Becker muscular dystrophy patients for deletions in 30 exons of the dystrophin gene by three-multiplex PCR

    SciTech Connect

    Risch, N. )

    1992-09-01

    Deletion mutations of the dystrophin gene may cause either the severe Duchenne muscular dystrophy (DMD) or the milder, allelic Becker muscular dystrophy (BMD) and are clustered in two high-frequency-deletion regions (HFDRs) located, respectively, 500 kb and 1,200 kb downstream from the 5[prime] end of the gene. Three PCR reactions described allowed the analysis of a total of 30 exons and led, to the identification of three additional deletions involving the following exons: (a) 42 only, (b) 28-42, and (c) 16 only, none of which were detected with the two original multiplex reactions. Therefore, the three modified multiplexes detected 95 of the 96 deletions identified among the 152 patients studied so far by using Southern analysis and cDNA probes. The only deletion that remained undetected with this system involves exons 22-25 and generates the junction fragment described elsewhere. The percentage of deletion mutations among DMS/BMD patients amounts to 63%, which is in agreement with similar estimates from other laboratories. When field-inversion gel electrophoresis is coupled to Southern analysis, the detection rate of deletion and duplication mutations reaches 65%.

  9. Preservation of Muscle Force in Mdx3cv Mice Correlates with Low-Level Expression of a Near Full-Length Dystrophin Protein

    PubMed Central

    Li, Dejia; Yue, Yongping; Duan, Dongsheng

    2008-01-01

    The complete absence of dystrophin causes Duchenne muscular dystrophy. Its restoration by greater than 20% is needed to reduce muscle pathology and improve muscle force. Dystrophin levels lower than 20% are considered therapeutically irrelevant but are associated with a less severe phenotype in certain Becker muscular dystrophy patients. To understand the role of low-level dystrophin expression, we compared muscle force and pathology in mdx3cv and mdx4cv mice. Dystrophin was eliminated in mdx4cv mouse muscle but was expressed in mdx3cv mice as a near full-length protein at ∼5% of normal levels. Consistent with previous reports, we found dystrophic muscle pathology in both mouse strains. Surprisingly, mdx3cv extensor digitorium longus muscle showed significantly higher tetanic force and was also more resistant to eccentric contraction-induced injury than mdx4cv extensor digitorium longus muscle. Furthermore, mdx3cv mice had stronger forelimb grip strength than mdx4cv mice. Immunostaining revealed utrophin up-regulation in both mouse strains. The dystrophin-associated glycoprotein complex was also restored in the sarcolemma in both strains although at levels lower than those in normal mice. Our results suggest that subtherapeutic expression levels of near full-length, membrane-bound dystrophin, possibly in conjunction with up-regulated utrophin levels, may help maintain minimal muscle force but not arrest muscle degeneration or necrosis. Our findings provide valuable insight toward understanding delayed clinical onset and/or slow disease progression in certain Becker muscular dystrophy patients. PMID:18385524

  10. 30 CFR 75.830 - Splicing and repair of trailing cables.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... including the replacement of: Insulation, semi-conductive tape, metallic shielding, and the outer jacket(s... include instructions for outer-jacket repairs and splices. (b) Splicing limitations. (1) Splicing of...

  11. 30 CFR 75.830 - Splicing and repair of trailing cables.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... including the replacement of: Insulation, semi-conductive tape, metallic shielding, and the outer jacket(s... include instructions for outer-jacket repairs and splices. (b) Splicing limitations. (1) Splicing of...

  12. Distribution of dystrophin- and utrophin-associated protein complexes (DAPC/UAPC) in human hematopoietic stem/progenitor cells.

    PubMed

    Teniente-De Alba, Carmen; Martínez-Vieyra, Ivette; Vivanco-Calixto, Raúl; Galván, Iván J; Cisneros, Bulmaro; Cerecedo, Doris

    2011-10-01

    Hematopoietic stem cells (HSC) are defined by their cardinal properties, such as sustained proliferation, multilineage differentiation, and self-renewal, which give rise to a hierarchy of progenitor populations with more restricted potential lineage, ultimately leading to the production of all types of mature blood cells. HSC are anchored by cell adhesion molecules to their specific microenvironment, thus regulating their cell cycle, while cell migration is essentially required for seeding the HSC of the fetal bone marrow (BM) during development as well as in adult BM homeostasis. The dystrophin-associated protein complex (DAPC) is a large group of membrane-associated proteins linking the cytoskeleton to the extracellular matrix and exhibiting scaffolding, adhesion, and signaling roles in muscle and non-muscle cells including mature blood cells. Because adhesion and migration are mechanisms that influence the fate of the HSC, we explored the presence and the feasible role of DAPC. In this study, we characterized the pattern expression by immunoblot technique and, by confocal microscopy analysis, the cellular distribution of dystrophin and utrophin gene products, and the dystrophin-associated proteins (α-, β-dystroglycan, α-syntrophin, α-dystrobrevin) in relation to actin filaments in freshly isolated CD34+ cells from umbilical cord blood. Immunoprecipitation assays demonstrated the presence of Dp71d/Dp71Δ110m ∼DAPC and Up400/Up140∼DAPC. The subcellular distribution of the two DAPC in actin-based structures suggests their dynamic participation in adhesion and cell migration. In addition, the particular protein pattern expression found in hematopoietic stem/progenitor cells might be indicative of their feasible participation during differentiation. PMID:21623922

  13. Evaluation of a 5-tier scheme proposed for classification of sequence variants using bioinformatic and splicing assay data: inter-reviewer variability and promotion of minimum reporting guidelines.

    PubMed

    Walker, Logan C; Whiley, Phillip J; Houdayer, Claude; Hansen, Thomas V O; Vega, Ana; Santamarina, Marta; Blanco, Ana; Fachal, Laura; Southey, Melissa C; Lafferty, Alan; Colombo, Mara; De Vecchi, Giovanna; Radice, Paolo; Spurdle, Amanda B

    2013-10-01

    Splicing assays are commonly undertaken in the clinical setting to assess the clinical relevance of sequence variants in disease predisposition genes. A 5-tier classification system incorporating both bioinformatic and splicing assay information was previously proposed as a method to provide consistent clinical classification of such variants. Members of the ENIGMA Consortium Splicing Working Group undertook a study to assess the applicability of the scheme to published assay results, and the consistency of classifications across multiple reviewers. Splicing assay data were identified for 235 BRCA1 and 176 BRCA2 unique variants, from 77 publications. At least six independent reviewers from research and/or clinical settings comprehensively examined splicing assay methods and data reported for 22 variant assays of 21 variants in four publications, and classified the variants using the 5-tier classification scheme. Inconsistencies in variant classification occurred between reviewers for 17 of the variant assays. These could be attributed to a combination of ambiguity in presentation of the classification criteria, differences in interpretation of the data provided, nonstandardized reporting of results, and the lack of quantitative data for the aberrant transcripts. We propose suggestions for minimum reporting guidelines for splicing assays, and improvements to the 5-tier splicing classification system to allow future evaluation of its performance as a clinical tool. PMID:23893897

  14. Definition of Proteasomal Peptide Splicing Rules for High-Efficiency Spliced Peptide Presentation by MHC Class I Molecules

    PubMed Central

    Berkers, Celia R.; de Jong, Annemieke; Schuurman, Karianne G.; Linnemann, Carsten; Meiring, Hugo D.; Janssen, Lennert; Neefjes, Jacques J.; Schumacher, Ton N. M.; Rodenko, Boris

    2015-01-01

    Peptide splicing, in which two distant parts of a protein are excised and then ligated to form a novel peptide, can generate unique MHC class I–restricted responses. Because these peptides are not genetically encoded and the rules behind proteasomal splicing are unknown, it is difficult to predict these spliced Ags. In the current study, small libraries of short peptides were used to identify amino acid sequences that affect the efficiency of this transpeptidation process. We observed that splicing does not occur at random, neither in terms of the amino acid sequences nor through random splicing of peptides from different sources. In contrast, splicing followed distinct rules that we deduced and validated both in vitro and in cells. Peptide ligation was quantified using a model peptide and demonstrated to occur with up to 30% ligation efficiency in vitro, provided that optimal structural requirements for ligation were met by both ligating partners. In addition, many splicing products could be formed from a single protein. Our splicing rules will facilitate prediction and detection of new spliced Ags to expand the peptidome presented by MHC class I Ags. PMID:26401003

  15. Screening the dystrophin gene suggests a high rate of polymorphism in general but no exonic deletions in schizophrenics

    SciTech Connect

    Lindor, N.M.; Sobell, J.L.; Thibodeau, S.N.

    1994-03-15

    The dystrophin gene, located at chromosome Xp21, was evaluated as a candidate gene in chronic schizophrenia in response to the report of a large family in which schizophrenia cosegregated with Becker muscular dystrophy. Genomic DNA from 94 men with chronic schizophrenia was evaluated by Southern blot analysis using cDNA probes that span exons 1-59. No exonic deletions were identified. An unexpectedly high rate of polymorphism was calculated in this study and two novel polymorphisms were found, demonstrating the usefulness of the candidate gene approach even when results of the original study are negative. 41 refs., 1 fig., 4 tabs.

  16. A spliced antigenic peptide comprising a single spliced amino acid is produced in the proteasome by reverse splicing of a longer peptide fragment followed by trimming.

    PubMed

    Michaux, Alexandre; Larrieu, Pierre; Stroobant, Vincent; Fonteneau, Jean-François; Jotereau, Francine; Van den Eynde, Benoît J; Moreau-Aubry, Agnès; Vigneron, Nathalie

    2014-02-15

    Peptide splicing is a novel mechanism of production of peptides relying on the proteasome and involving the linkage of fragments originally distant in the parental protein. Peptides produced by splicing can be presented on class I molecules of the MHC and recognized by CTLs. In this study, we describe a new antigenic peptide, which is presented by HLA-A3 and comprises two noncontiguous fragments of the melanoma differentiation Ag gp100(PMEL17) spliced together in the reverse order to that in which they appear in the parental protein. Contrary to the previously described spliced peptides, which are produced by the association of fragments of 3-6 aa, the peptide described in this work results from the ultimate association of an 8-aa fragment with a single arginine residue. As described before, peptide splicing takes place in the proteasome by transpeptidation involving an acyl-enzyme intermediate linking one of the peptide fragment to a catalytic subunit of the proteasome. Interestingly, we observe that the peptide causing the nucleophilic attack on the acyl-enzyme intermediate must be at least 3 aa long to give rise to a spliced peptide. The spliced peptide produced from this reaction therefore bears an extended C terminus that needs to be further trimmed to produce the final antigenic peptide. We show that the proteasome is able to perform the final trimming step required to produce the antigenic peptide described in this work. PMID:24453253

  17. Altered astrocyte morphology and vascular development in dystrophin-Dp71-null mice.

    PubMed

    Giocanti-Auregan, Audrey; Vacca, Ophélie; Bénard, Romain; Cao, Sijia; Siqueiros, Lourdes; Montañez, Cecilia; Paques, Michel; Sahel, José-Alain; Sennlaub, Florian; Guillonneau, Xavier; Rendon, Alvaro; Tadayoni, Ramin

    2016-05-01

    Understanding retinal vascular development is crucial because many retinal vascular diseases such as diabetic retinopathy (in adults) or retinopathy of prematurity (in children) are among the leading causes of blindness. Given the localization of the protein Dp71 around the retinal vessels in adult mice and its role in maintaining retinal homeostasis, the aim of this study was to determine if Dp71 was involved in astrocyte and vascular development regulation. An experimental study in mouse retinas was conducted. Using a dual immunolabeling with antibodies to Dp71 and anti-GFAP for astrocytes on retinal sections and isolated astrocytes, it was found that Dp71 was expressed in wild-type (WT) mouse astrocytes from early developmental stages to adult stage. In Dp71-null mice, a reduction in GFAP-immunopositive astrocytes was observed as early as postnatal day 6 (P6) compared with WT mice. Using real-time PCR, it was showed that Dp71 mRNA was stable between P1 and P6, in parallel with post-natal vascular development. Regarding morphology in Dp71-null and WT mice, a significant decrease in overall astrocyte process number in Dp71-null retinas at P6 to adult age was found. Using fluorescence-conjugated isolectin Griffonia simplicifolia on whole mount retinas, subsequent delay of developing vascular network at the same age in Dp71-null mice was found. An evidence that the Dystrophin Dp71, a membrane-associated cytoskeletal protein and one of the smaller Duchenne muscular dystrophy gene products, regulates astrocyte morphology and density and is associated with subsequent normal blood vessel development was provided. GLIA 2016;64:716-729. PMID:26711882

  18. Effects of Dantrolene Therapy on Disease Phenotype in Dystrophin Deficient mdx Mice.

    PubMed

    Quinn, James L; Huynh, Tony; Uaesoontrachoon, Kitipong; Tatem, Kathleen; Phadke, Aditi; Van der Meulen, Jack H; Yu, Qing; Nagaraju, Kannaboyina

    2013-01-01

    Dystrophin deficiency causes contraction-induced injury and damage to the muscle fiber, resulting in sustained increase in intracellular calcium levels, activation of calcium-dependent proteases and cell death. It is known that the Ryanodine receptor (RyR1) on the sarcoplasmic reticular (SR) membrane controls calcium release. Dantrolene, an FDA approved skeletal muscle relaxant, inhibits the release of calcium from the SR during excitation-contraction and suppresses uncontrolled calcium release by directly acting on the RyR complex to limit its activation. This study examines whether Dantrolene can reduce the disease phenotype in the mdx mouse model of muscular dystrophy. We treated mdx mice (4 weeks old) with daily intraperitoneal injections of 40mg/kg of Dantrolene for 6 weeks and measured functional (grip strength, in vitro force contractions), behavioral (open field digiscan), imagining (optical imaging for inflammation), histological (H&E), and molecular (protein and RNA) endpoints in a blinded fashion. We found that treatment with Dantrolene resulted in decreased grip strength and open field behavioral activity in mdx mice. There was no significant difference in inflammation either by optical imaging analysis of cathepsin activity or histological (H&E) analysis. In vitro force contraction measures showed no changes in EDL muscle-specific force, lengthening-contraction force deficit, or fatigue resistance. We found Dantrolene treatment significantly reduces serum CK levels. Further, Dantrolene-treated mice showed decreased SERCA1 but not RyR1 expression in skeletal muscle. These results suggest that Dantrolene treatment alone has no significant beneficial effects at the tested doses in young mdx mice. PMID:24270550

  19. Effects of Dantrolene Therapy on Disease Phenotype in Dystrophin Deficient mdx Mice

    PubMed Central

    Quinn, James L; Huynh, Tony; Uaesoontrachoon, Kitipong; Tatem, Kathleen; Phadke, Aditi; Van der Meulen, Jack H; Yu, Qing; Nagaraju, Kannaboyina

    2013-01-01

    Dystrophin deficiency causes contraction-induced injury and damage to the muscle fiber, resulting in sustained increase in intracellular calcium levels, activation of calcium-dependent proteases and cell death. It is known that the Ryanodine receptor (RyR1) on the sarcoplasmic reticular (SR) membrane controls calcium release. Dantrolene, an FDA approved skeletal muscle relaxant, inhibits the release of calcium from the SR during excitation-contraction and suppresses uncontrolled calcium release by directly acting on the RyR complex to limit its activation. This study examines whether Dantrolene can reduce the disease phenotype in the mdx mouse model of muscular dystrophy. We treated mdx mice (4 weeks old) with daily intraperitoneal injections of 40mg/kg of Dantrolene for 6 weeks and measured functional (grip strength, in vitro force contractions), behavioral (open field digiscan), imagining (optical imaging for inflammation), histological (H&E), and molecular (protein and RNA) endpoints in a blinded fashion. We found that treatment with Dantrolene resulted in decreased grip strength and open field behavioral activity in mdx mice. There was no significant difference in inflammation either by optical imaging analysis of cathepsin activity or histological (H&E) analysis. In vitro force contraction measures showed no changes in EDL muscle-specific force, lengthening-contraction force deficit, or fatigue resistance. We found Dantrolene treatment significantly reduces serum CK levels. Further, Dantrolene-treated mice showed decreased SERCA1 but not RyR1 expression in skeletal muscle. These results suggest that Dantrolene treatment alone has no significant beneficial effects at the tested doses in young mdx mice. PMID:24270550

  20. Disruption of action potential and calcium signaling properties in malformed myofibers from dystrophin-deficient mice

    PubMed Central

    Hernández-Ochoa, Erick O; Pratt, Stephen J P; Garcia-Pelagio, Karla P; Schneider, Martin F; Lovering, Richard M

    2015-01-01

    Duchenne muscular dystrophy (DMD), the most common and severe muscular dystrophy, is caused by the absence of dystrophin. Muscle weakness and fragility (i.e., increased susceptibility to damage) are presumably due to structural instability of the myofiber cytoskeleton, but recent studies suggest that the increased presence of malformed/branched myofibers in dystrophic muscle may also play a role. We have previously studied myofiber morphology in healthy wild-type (WT) and dystrophic (MDX) skeletal muscle. Here, we examined myofiber excitability using high-speed confocal microscopy and the voltage-sensitive indicator di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS) to assess the action potential (AP) properties. We also examined AP-induced Ca2+ transients using high-speed confocal microscopy with rhod-2, and assessed sarcolemma fragility using elastimetry. AP recordings showed an increased width and time to peak in malformed MDX myofibers compared to normal myofibers from both WT and MDX, but no significant change in AP amplitude. Malformed MDX myofibers also exhibited reduced AP-induced Ca2+ transients, with a further Ca2+ transient reduction in the branches of malformed MDX myofibers. Mechanical studies indicated an increased sarcolemma deformability and instability in malformed MDX myofibers. The data suggest that malformed myofibers are functionally different from myofibers with normal morphology. The differences seen in AP properties and Ca2+ signals suggest changes in excitability and remodeling of the global Ca2+ signal, both of which could underlie reported weakness in dystrophic muscle. The biomechanical changes in the sarcolemma support the notion that malformed myofibers are more susceptible to damage. The high prevalence of malformed myofibers in dystrophic muscle may contribute to the progressive strength loss and fragility seen in dystrophic muscles. PMID:25907787

  1. Biomedical Impact of Splicing Mutations Revealed through Exome Sequencing

    PubMed Central

    Taneri, Bahar; Asilmaz, Esra; Gaasterland, Terry

    2012-01-01

    Splicing is a cellular mechanism, which dictates eukaryotic gene expression by removing the noncoding introns and ligating the coding exons in the form of a messenger RNA molecule. Alternative splicing (AS) adds a major level of complexity to this mechanism and thus to the regulation of gene expression. This widespread cellular phenomenon generates multiple messenger RNA isoforms from a single gene, by utilizing alternative splice sites and promoting different exon–intron inclusions and exclusions. AS greatly increases the coding potential of eukaryotic genomes and hence contributes to the diversity of eukaryotic proteomes. Mutations that lead to disruptions of either constitutive splicing or AS cause several diseases, among which are myotonic dystrophy and cystic fibrosis. Aberrant splicing is also well established in cancer states. Identification of rare novel mutations associated with splice-site recognition, and splicing regulation in general, could provide further insight into genetic mechanisms of rare diseases. Here, disease relevance of aberrant splicing is reviewed, and the new methodological approach of starting from disease phenotype, employing exome sequencing and identifying rare mutations affecting splicing regulation is described. Exome sequencing has emerged as a reliable method for finding sequence variations associated with various disease states. To date, genetic studies using exome sequencing to find disease-causing mutations have focused on the discovery of nonsynonymous single nucleotide polymorphisms that alter amino acids or introduce early stop codons, or on the use of exome sequencing as a means to genotype known single nucleotide polymorphisms. The involvement of splicing mutations in inherited diseases has received little attention and thus likely occurs more frequently than currently estimated. Studies of exome sequencing followed by molecular and bioinformatic analyses have great potential to reveal the high impact of splicing

  2. Altered PLP1 splicing causes hypomyelination of early myelinating structures

    PubMed Central

    Kevelam, Sietske H; Taube, Jennifer R; van Spaendonk, Rosalina M L; Bertini, Enrico; Sperle, Karen; Tarnopolsky, Mark; Tonduti, Davide; Valente, Enza Maria; Travaglini, Lorena; Sistermans, Erik A; Bernard, Geneviève; Catsman-Berrevoets, Coriene E; van Karnebeek, Clara D M; Østergaard, John R; Friederich, Richard L; Fawzi Elsaid, Mahmoud; Schieving, Jolanda H; Tarailo-Graovac, Maja; Orcesi, Simona; Steenweg, Marjan E; van Berkel, Carola G M; Waisfisz, Quinten; Abbink, Truus E M; van der Knaap, Marjo S; Hobson, Grace M; Wolf, Nicole I

    2015-01-01

    Objective The objective of this study was to investigate the genetic etiology of the X-linked disorder “Hypomyelination of Early Myelinating Structures” (HEMS). Methods We included 16 patients from 10 families diagnosed with HEMS by brain MRI criteria. Exome sequencing was used to search for causal mutations. In silico analysis of effects of the mutations on splicing and RNA folding was performed. In vitro gene splicing was examined in RNA from patients’ fibroblasts and an immortalized immature oligodendrocyte cell line after transfection with mutant minigene splicing constructs. Results All patients had unusual hemizygous mutations of PLP1 located in exon 3B (one deletion, one missense and two silent), which is spliced out in isoform DM20, or in intron 3 (five mutations). The deletion led to truncation of PLP1, but not DM20. Four mutations were predicted to affect PLP1/DM20 alternative splicing by creating exonic splicing silencer motifs or new splice donor sites or by affecting the local RNA structure of the PLP1 splice donor site. Four deep intronic mutations were predicted to destabilize a long-distance interaction structure in the secondary PLP1 RNA fragment involved in regulating PLP1/DM20 alternative splicing. Splicing studies in fibroblasts and transfected cells confirmed a decreased PLP1/DM20 ratio. Interpretation Brain structures that normally myelinate early are poorly myelinated in HEMS, while they are the best myelinated structures in Pelizaeus–Merzbacher disease, also caused by PLP1 alterations. Our data extend the phenotypic spectrum of PLP1-related disorders indicating that normal PLP1/DM20 alternative splicing is essential for early myelination and support the need to include intron 3 in diagnostic sequencing. PMID:26125040

  3. An artificial riboswitch for controlling pre-mRNA splicing.

    PubMed

    Kim, Dong-Suk; Gusti, Veronica; Pillai, Sailesh G; Gaur, Rajesh K

    2005-11-01

    Riboswitches, as previously reported, are natural RNA aptamers that regulate the expression of numerous bacterial metabolic genes in response to small molecule ligands. It has recently been shown that these RNA genetic elements are also present near the splice site junctions of plant and fungal introns, thus raising the possibility of their involvement in regulating mRNA splicing. Here it is shown for the first time that a riboswitch can be engineered to regulate pre-mRNA splicing in vitro. We show that insertion of a high-affinity theophylline binding aptamer into the 3' splice site (3' ss) region of a model pre-mRNA (AdML-Theo29AG) enables its splicing to be repressed by the addition theophylline. Our results indicate that the location of 3' ss AG within the aptamer plays a crucial role in conferring theophylline-dependent control of pre-mRNA splicing. We also show that theophylline-mediated control of pre-mRNA splicing is highly specific by first demonstrating that a small molecule ligand similar in shape and size to theophylline had no effect on the splicing of AdML-Theo29AG pre-mRNA. Second, theophylline failed to exert any influence on the splicing of a pre-mRNA that does not contain its binding site. Third, theophylline specifically blocks the step II of the splicing reaction. Finally, we provide evidence that theophylline-dependent control of pre-mRNA splicing is functionally relevant. PMID:16244133

  4. Modulation of RNA splicing as a potential treatment for cancer.

    PubMed

    Bauman, John A; Kole, Ryszard

    2011-01-01

    Close to 90% of human genes are transcribed into pre-mRNA that undergoes alternative splicing, producing multiple mRNAs and proteins from single genes. This process is largely responsible for human proteome diversity, and about half of genetic disease-causing mutations affect splicing. Splice-switching oligonucleotides (SSOs) comprise an emerging class of antisense therapeutics that modify gene expression by directing pre-mRNA splice site usage. Bauman et al. investigated an SSO that up-regulated the expression of an anti-cancer splice variant while simultaneously eliminating an over-expressed cancer-causing splice variant.  This was accomplished by targeting pre-mRNA of the apoptotic regulator Bcl-x, which is alternatively spliced to express anti- and pro-apoptotic splice variants Bcl-xL and Bcl-xS, respectively. High expression of Bcl-xL is a hallmark of many cancers and is considered a general mechanism used by cancer cells to evade apoptosis. Redirection of Bcl-x pre-mRNA splicing from Bcl-xL to -xS by SSO induced apoptotic and chemosensitizing effects in various cancer cell lines. Importantly, the paper shows that delivery of Bcl-x SSO using a lipid nanoparticle redirected Bcl-x splicing and reduced tumor burden in melanoma lung metastases. This was the first demonstration of SSO efficacy in tumors in vivo. SSOs are not limited to be solely potential anti-cancer drugs. SSOs were first applied to repair aberrant splicing in thalassemia, a genetic disease, they have been used to create novel proteins (e.g., ∆7TNFR1), and they have recently progressed to clinical trials for patients with Duchenne muscular dystrophy.  PMID:21637003

  5. Spliced DNA Sequences in the Paramecium Germline: Their Properties and Evolutionary Potential

    PubMed Central

    Catania, Francesco; McGrath, Casey L.; Doak, Thomas G.; Lynch, Michael

    2013-01-01

    Despite playing a crucial role in germline-soma differentiation, the evolutionary significance of developmentally regulated genome rearrangements (DRGRs) has received scant attention. An example of DRGR is DNA splicing, a process that removes segments of DNA interrupting genic and/or intergenic sequences. Perhaps, best known for shaping immune-system genes in vertebrates, DNA splicing plays a central role in the life of ciliated protozoa, where thousands of germline DNA segments are eliminated after sexual reproduction to regenerate a functional somatic genome. Here, we identify and chronicle the properties of 5,286 sequences that putatively undergo DNA splicing (i.e., internal eliminated sequences [IESs]) across the genomes of three closely related species of the ciliate Paramecium (P. tetraurelia, P. biaurelia, and P. sexaurelia). The study reveals that these putative IESs share several physical characteristics. Although our results are consistent with excision events being largely conserved between species, episodes of differential IES retention/excision occur, may have a recent origin, and frequently involve coding regions. Our findings indicate interconversion between somatic—often coding—DNA sequences and noncoding IESs, and provide insights into the role of DNA splicing in creating potentially functional genetic innovation. PMID:23737328

  6. Single-molecule RNA observation in vivo reveals dynamics of co-transcriptional splicing

    NASA Astrophysics Data System (ADS)

    Ferguson, M. L.; Coulon, A.; de Turris, V.; Palangat, M.; Chow, C. C.; Singer, R. H.; Larson, D. R.

    2013-03-01

    The synthesis of pre-mRNA and the splicing of that pre-mRNA to form completed transcripts requires coordination between two large multi-subunit complexes (the transcription elongation complex and the spliceosome). How this coordination occurs in vivo is unknown. Here we report the first experimental observation of transcription and splicing occurring at the same gene in living cells. By utilizing the PP7/MS2 fluorescent RNA reporter system, we can directly observe two distinct regions of the nascent RNA, allowing us to measure the rise and fall time of the intron and exon of a reporter gene stably integrated into a human cell line. The reporter gene consists of a beta globin gene where we have inserted a 24 RNA hairpin cassette into the intron/exon. Upon synthesis, the RNA hairpins are tightly bound by fluorescently-labeled PP7/MS2 bacteriophage coat proteins. After gene induction, a single locus of active transcription in the nucleus shows fluorescence intensity changes characteristic of the synthesis and excision of the intron/exon. Using fluctuation analysis, we determine the elongation rate to be 1.5 kb/min. From the temporal cross correlation function, we determine that splicing of this gene must be co-transcriptional with a splicing time of ~100 seconds before termination and a ~200 second pause at termination. We propose that dual-color RNA imaging may be extended to investigate other mechanisms of transcription, gene regulation, and RNA processing.

  7. Quantitative Profiling of Drosophila melanogaster Dscam1 Isoforms Reveals No Changes in Splicing after Bacterial Exposure

    PubMed Central

    Kurtz, Joachim; Schmucker, Dietmar; Chen, Wei

    2014-01-01

    The hypervariable Dscam1 (Down syndrome cell adhesion molecule 1) gene can produce thousands of different ectodomain isoforms via mutually exclusive alternative splicing. Dscam1 appears to be involved in the immune response of some insects and crustaceans. It has been proposed that the diverse isoforms may be involved in the recognition of, or the defence against, diverse parasite epitopes, although evidence to support this is sparse. A prediction that can be generated from this hypothesis is that the gene expression of specific exons and/or isoforms is influenced by exposure to an immune elicitor. To test this hypothesis, we for the first time, use a long read RNA sequencing method to directly investigate the Dscam1 splicing pattern after exposing adult Drosophila melanogaster and a S2 cell line to live Escherichia coli. After bacterial exposure both models showed increased expression of immune-related genes, indicating that the immune system had been activated. However there were no changes in total Dscam1 mRNA expression. RNA sequencing further showed that there were no significant changes in individual exon expression and no changes in isoform splicing patterns in response to bacterial exposure. Therefore our studies do not support a change of D. melanogaster Dscam1 isoform diversity in response to live E. coli. Nevertheless, in future this approach could be used to identify potentially immune-related Dscam1 splicing regulation in other host species or in response to other pathogens. PMID:25310676

  8. Opioid inhibition of N-type Ca2+ channels and spinal analgesia couple to alternative splicing.

    PubMed

    Andrade, Arturo; Denome, Sylvia; Jiang, Yu-Qiu; Marangoudakis, Spiro; Lipscombe, Diane

    2010-10-01

    Alternative pre-mRNA splicing occurs extensively in the nervous systems of complex organisms, including humans, considerably expanding the potential size of the proteome. Cell-specific alternative pre-mRNA splicing is thought to optimize protein function for specialized cellular tasks, but direct evidence for this is limited. Transmission of noxious thermal stimuli relies on the activity of N-type Ca(V)2.2 calcium channels in nociceptors. Using an exon-replacement strategy in mice, we show that mutually exclusive splicing patterns in the Ca(V)2.2 gene modulate N-type channel function in nociceptors, leading to a change in morphine analgesia. Exon 37a (e37a) enhances μ-opioid receptor-mediated inhibition of N-type calcium channels by promoting activity-independent inhibition. In the absence of e37a, spinal morphine analgesia is weakened in vivo but the basal response to noxious thermal stimuli is not altered. Our data suggest that highly specialized, discrete cellular responsiveness in vivo can be attributed to alternative splicing events regulated at the level of individual neurons. PMID:20852623

  9. Probing the interplay between the two steps of group I intron splicing: competition of exogenous guanosine with omega G.

    PubMed

    Zarrinkar, P P; Sullenger, B A

    1998-12-22

    One largely unexplored question about group I intron splicing is how the cleavage and ligation steps of the reaction are coordinated. We describe a simple in vitro trans-splicing model system in which both steps take place, including the exchange of ligands in the guanosine-binding site that must occur between the two steps. Using this model system, we show that the switch is accomplished by modulating the relative affinity of the binding site for the two ligands. While the terminal guanosine of the intron (omegaG) and exogenous guanosine compete for binding during the first step of splicing, no competition is apparent during the second step, when omegaG is bound tightly. These results help explain how the ribozyme orchestrates progression through the splicing reaction. In addition to providing a new tool to ask basic questions about RNA catalysis, the trans-splicing model system will also facilitate the development of therapeutically useful group I ribozymes that can repair mutant mRNAs. PMID:9922174

  10. Sporadic ALS has compartment-specific aberrant exon splicing and altered cell–matrix adhesion biology

    PubMed Central

    Rabin, Stuart J.; Kim, Jae Mun ‘Hugo’; Baughn, Michael; Libby, Ryan T.; Kim, Young Joo; Fan, Yuxin; Libby, Randell T.; La Spada, Albert; Stone, Brad; Ravits, John

    2010-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive weakness from loss of motor neurons. The fundamental pathogenic mechanisms are unknown and recent evidence is implicating a significant role for abnormal exon splicing and RNA processing. Using new comprehensive genomic technologies, we studied exon splicing directly in 12 sporadic ALS and 10 control lumbar spinal cords acquired by a rapid autopsy system that processed nervous systems specifically for genomic studies. ALS patients had rostral onset and caudally advancing disease and abundant residual motor neurons in this region. We created two RNA pools, one from motor neurons collected by laser capture microdissection and one from the surrounding anterior horns. From each, we isolated RNA, amplified mRNA, profiled whole-genome exon splicing, and applied advanced bioinformatics. We employed rigorous quality control measures at all steps and validated findings by qPCR. In the motor neuron enriched mRNA pool, we found two distinct cohorts of mRNA signals, most of which were up-regulated: 148 differentially expressed genes (P ≤ 10−3) and 411 aberrantly spliced genes (P ≤ 10−5). The aberrantly spliced genes were highly enriched in cell adhesion (P ≤ 10−57), especially cell–matrix as opposed to cell–cell adhesion. Most of the enriching genes encode transmembrane or secreted as opposed to nuclear or cytoplasmic proteins. The differentially expressed genes were not biologically enriched. In the anterior horn enriched mRNA pool, we could not clearly identify mRNA signals or biological enrichment. These findings, perturbed and up-regulated cell–matrix adhesion, suggest possible mechanisms for the contiguously progressive nature of motor neuron degeneration. Data deposition: GeneChip raw data (CEL-files) have been deposited for public access in the Gene Expression Omnibus (GEO), www.ncbi.nlm.nih.gov/geo, accession number GSE18920. PMID:19864493

  11. Low frequency ultrasonic nondestructive inspection of aluminum/adhesive fuselage lap splices

    SciTech Connect

    Patton, T.

    1994-01-04

    This thesis is a collection of research efforts in ultrasonics, conducted at the Center for Aviation Systems Reliability located at Iowa State University, as part of the Federal Aviation Administration`s ``Aging Aircraft Program.`` The research was directed toward the development of an ultrasonic prototype to inspect the aluminum/adhesive fuselage lap splices found on 1970`s vintage Boeing passenger aircraft. The ultrasonic prototype consists of a normal incidence, low frequency inspection technique, and a scanning adapter that allows focused immersion transducers to be operated in a direct contact manner in any inspection orientation, including upside-down. The inspection technique uses a computer-controlled data acquisition system to produce a C-scan image of a radio frequency (RF) waveform created by a low frequency, broadband, focused beam transducer, driven with a spike voltage pulser. C-scans produced by this technique are color representations of the received signal`s peak-to-peak amplitude (voltage) taken over an (x, y) grid. Low frequency, in this context, refers to a wavelength that is greater than the lap splice`s layer thicknesses. With the low frequency technique, interface echoes of the lap splice are not resolved and gating of the signal is unnecessary; this in itself makes the technique simple to implement and saves considerable time in data acquisition. Along with the advantages in data acquisition, the low frequency technique is relatively insensitive to minor surface curvature and to ultrasonic interference effects caused by adhesive bondline thickness variations in the lap splice.

  12. C-Terminal-Truncated Microdystrophin Recruits Dystrobrevin and Syntrophin to the Dystrophin-Associated Glycoprotein Complex and Reduces Muscular Dystrophy in Symptomatic Utrophin/Dystrophin Double-Knockout Mice

    PubMed Central

    Yue, Yongping; Liu, Mingju; Duan, Dongsheng

    2007-01-01

    C-terminal-truncated (ΔC) microdystrophin is being developed for Duchenne muscular dystrophy gene therapy. Encouraging results have been achieved in the mdx mouse model. Unfortunately, mdx mice do not display the same phenotype as human patients. Evaluating ΔC microdystrophin in a symptomatic model will be of significant relevance to human trials. Utrophin/dystrophin double-knockout (u-dko) mice were developed to model severe dystrophic changes in human patients. In this study we evaluated the therapeutic effect of the ΔR4-R23/ΔC microdystrophin gene (ΔR4/ΔC) after serotype-6 adeno-associated virus-mediated gene transfer in neonatal u-dko muscle. At 2 months after gene transfer, the percentage of centrally nucleated myofiber was reduced from 89.2 to 3.4% and muscle weight was normalized. Furthermore, we have demonstrated for the first time that ΔC microdystrophin can eliminate interstitial fibrosis and macrophage infiltration and restore dystrobrevin and syntrophin to the dystrophin-associated glycoprotein complex. Interestingly neuronal nitric oxide synthase was not restored. The most impressive results were achieved in muscle force measurement. Neonatal gene therapy increased twitch- and tetanic-specific force. It also brought the response to eccentric contraction-induced injury to the normal range. In summary, our results suggest that the ΔR4/ΔC microgene holds great promise in preventing muscular dystrophy. PMID:16563874

  13. Regulation of alternative splicing of liver scavenger receptor class B gene by estrogen and the involved regulatory splicing factors.

    PubMed

    Zhang, Xiaohui; Moor, Andrea N; Merkler, Kathleen A; Liu, Qiyuan; McLean, Mark P

    2007-11-01

    The scavenger receptor class B isoforms (SR-B) type I and type II mediate the selective uptake of high-density lipoprotein cholesterol and promote reverse cholesterol transport, an important atherosclerosis protection mechanism, in the liver. Previously it was shown that the hepatic expression of SR-BI and SR-BII is regulated by estrogen. In the present study, we demonstrate that estrogen differentially regulates expression of the glycosylated and nonglycosylated forms of SR-BI and SR-BII in rat liver and hepatic cells. We report that estrogen mainly induces the down-regulation of glycosylated SR-BI and the up-regulation of nonglycosylated SR-BII. To study how estrogen regulates expression of the SR-B isoforms, we constructed a SR-B minigene containing minimal genomic sequences and were able to demonstrate that estrogen directly regulates the pre-mRNA alternative splicing of the exogenously expressed SR-B minigene in hepatic cells. Furthermore, we showed that the overexpression of splicing factors alternative splicing factor/splicing factor 2, Transformer (Tra)-2alpha, and Tra2beta changes the splicing pattern of SR-B dramatically, whereas other splicing factors, such as heterogeneous nuclear ribonucleoprotein-G, SC-35, and arginine/serine-rich p40, had no effect. We also demonstrate that estrogen regulates Tra2beta expression levels in liver cells. These studies suggest that estrogen may regulate SR-B isoform expression at both the RNA splicing and posttranslational modification levels and that, for alternative splicing regulation, estrogen may function by regulating the expression of the splicing factors alternative splicing factor/splicing factor 2, Tra2alpha, and especially Tra2beta. PMID:17673517

  14. Connecting the dots: chromatin and alternative splicing in EMT

    PubMed Central

    Warns, Jessica A.; Davie, James R.; Dhasarathy, Archana

    2015-01-01

    Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process. PMID:26291837

  15. Identification of a new splice variant of BDNF in chicken

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Brain-derived neurotrophic factor (BDNF) appears to be involved in the central regulation of energy homeostasis. BDNF splicing variants were discovered in vertebrates. Results from human, mouse and rat suggest that alternative BDNF splicing variants potentially play a role in fat deposition. Using t...

  16. Alternative splicing of inner-ear-expressed genes.

    PubMed

    Wang, Yanfei; Liu, Yueyue; Nie, Hongyun; Ma, Xin; Xu, Zhigang

    2016-09-01

    Alternative splicing plays a fundamental role in the development and physiological function of the inner ear. Inner-ear-specific gene splicing is necessary to establish the identity and maintain the function of the inner ear. For example, exon 68 of Cadherin 23 (Cdh23) gene is subject to inner-ear-specific alternative splicing, and as a result, Cdh23(+ 68) is only expressed in inner ear hair cells. Alternative splicing along the tonotopic axis of the cochlea contributes to frequency tuning, particularly in lower vertebrates, such as chickens and turtles. Differential splicing of Kcnma1, which encodes for the α subunit of the Ca(2+)-activated K(+) channel (BK channel), has been suggested to affect the channel gating properties and is important for frequency tuning. Consequently, deficits in alternative splicing have been shown to cause hearing loss, as we can observe in Bronx Waltzer (bv) mice and Sfswap mutant mice. Despite the advances in this field, the regulation of alternative splicing in the inner ear remains elusive. Further investigation is also needed to clarify the mechanism of hearing loss caused by alternative splicing deficits. PMID:27376950

  17. Alternative Splicing of Pre-mRNA in Cancer

    PubMed Central

    Körner, Meike; Miller, Laurence J.

    2009-01-01

    Through alternative splicing, multiple different transcripts can be generated from a single gene. Alternative splicing represents an important molecular mechanism of gene regulation in physiological processes such as developmental programming as well as in disease. In cancer, splicing is significantly altered. Tumors express a different collection of alternative spliceoforms than normal tissues. Many tumor-associated splice variants arise from genes with an established role in carcinogenesis or tumor progression, and their functions can be oncogenic. This raises the possibility that products of alternative splicing play a pathogenic role in cancer. Moreover, cancer-associated spliceoforms represent potential diagnostic biomarkers and therapeutic targets. G protein-coupled peptide hormone receptors provide a good illustration of alternative splicing in cancer. The wild-type forms of these receptors have long been known to be expressed in cancer and to modulate tumor cell functions. They are also recognized as attractive clinical targets. Recently, splice variants of these receptors have been increasingly identified in various types of cancer. In particular, alternative cholecystokinin type 2, secretin, and growth hormone-releasing hormone receptor spliceoforms are expressed in tumors. Peptide hormone receptor splice variants can fundamentally differ from their wild-type receptor counterparts in pharmacological and functional characteristics, in their distribution in normal and malignant tissues, and in their potential use for clinical applications. PMID:19574427

  18. Involvement of PARP1 in the regulation of alternative splicing

    PubMed Central

    Matveeva, Elena; Maiorano, John; Zhang, Qingyang; Eteleeb, Abdallah M; Convertini, Paolo; Chen, Jing; Infantino, Vittoria; Stamm, Stefan; Wang, Jiping; Rouchka, Eric C; Fondufe-Mittendorf, Yvonne N

    2016-01-01

    Specialized chromatin structures such as nucleosomes with specific histone modifications decorate exons in eukaryotic genomes, suggesting a functional connection between chromatin organization and the regulation of pre-mRNA splicing. Through profiling the functional location of Poly (ADP) ribose polymerase, we observed that it is associated with the nucleosomes at exon/intron boundaries of specific genes, suggestive of a role for this enzyme in alternative splicing. Poly (ADP) ribose polymerase has previously been implicated in the PARylation of splicing factors as well as regulation of the histone modification H3K4me3, a mark critical for co-transcriptional splicing. In light of these studies, we hypothesized that interaction of the chromatin-modifying factor, Poly (ADP) ribose polymerase with nucleosomal structures at exon–intron boundaries, might regulate pre-mRNA splicing. Using genome-wide approaches validated by gene-specific assays, we show that depletion of PARP1 or inhibition of its PARylation activity results in changes in alternative splicing of a specific subset of genes. Furthermore, we observed that PARP1 bound to RNA, splicing factors and chromatin, suggesting that Poly (ADP) ribose polymerase serves as a gene regulatory hub to facilitate co-transcriptional splicing. These studies add another function to the multi-functional protein, Poly (ADP) ribose polymerase, and provide a platform for further investigation of this protein’s function in organizing chromatin during gene regulatory processes. PMID:27462443

  19. Global analysis of alternative splicing differences between humans and chimpanzees.

    PubMed

    Calarco, John A; Xing, Yi; Cáceres, Mario; Calarco, Joseph P; Xiao, Xinshu; Pan, Qun; Lee, Christopher; Preuss, Todd M; Blencowe, Benjamin J

    2007-11-15

    Alternative splicing is a powerful mechanism affording extensive proteomic and regulatory diversity from a limited repertoire of genes. However, the extent to which alternative splicing has contributed to the evolution of primate species-specific characteristics has not been assessed previously. Using comparative genomics and quantitative microarray profiling, we performed the first global analysis of alternative splicing differences between humans and chimpanzees. Surprisingly, 6%-8% of profiled orthologous exons display pronounced splicing level differences in the corresponding tissues from the two species. Little overlap is observed between the genes associated with alternative splicing differences and the genes that display steady-state transcript level differences, indicating that these layers of regulation have evolved rapidly to affect distinct subsets of genes in humans and chimpanzees. The alternative splicing differences we detected are predicted to affect diverse functions including gene expression, signal transduction, cell death, immune defense, and susceptibility to diseases. Differences in expression at the protein level of the major splice variant of Glutathione S-transferase omega-2 (GSTO2), which functions in the protection against oxidative stress and is associated with human aging-related diseases, suggests that this enzyme is less active in human cells compared with chimpanzee cells. The results of this study thus support an important role for alternative splicing in establishing differences between humans and chimpanzees. PMID:17978102

  20. Discovering Transcription and Splicing Networks in Myelodysplastic Syndromes

    PubMed Central

    Wang, Hongyan; Wen, Jianguo; Chang, Chung-che; Zhou, Xiaobo

    2013-01-01

    More and more transcription factors and their motifs have been reported and linked to specific gene expression levels. However, focusing only on transcription is not sufficient for mechanism research. Most genes, especially in eukaryotes, are alternatively spliced to different isoforms. Some of these isoforms increase the biodiversity of proteins. From this viewpoint, transcription and splicing are two of important mechanisms to modulate expression levels of isoforms. To integrate these two kinds of regulation, we built a linear regression model to select a subset of transcription factors and splicing factors for each co-expressed isoforms using least-angle regression approach. Then, we applied this method to investigate the mechanism of myelodysplastic syndromes (MDS), a precursor lesion of acute myeloid leukemia. Results suggested that expression levels of most isoforms were regulated by a set of selected regulatory factors. Some of the detected factors, such as EGR1 and STAT family, are highly correlated with progression of MDS. We discovered that the splicing factor SRSF11 experienced alternative splicing switch, and in turn induced different amino acid sequences between MDS and controls. This splicing switch causes two different splicing mechanisms. Polymerase Chain Reaction experiments also confirmed that one of its isoforms was over-expressed in MDS. We analyzed the regulatory networks constructed from the co-expressed isoforms and their regulatory factors in MDS. Many of these networks were enriched in the herpes simplex infection pathway which involves many splicing factors, and pathways in cancers and acute or chronic myeloid leukemia. PMID:24244432

  1. Splice Form Dependence of b-Neurexin/Neuroligin Binding Interactions

    SciTech Connect

    Koehnke, J.; Katsamba, P; Ahlsen, G; Bahna, F; Vendome, J; Honig, B; Shapiro, L; Jin, X

    2010-01-01

    Alternatively spliced {beta}-neurexins ({beta}-NRXs) and neuroligins (NLs) are thought to have distinct extracellular binding affinities, potentially providing a {beta}-NRX/NL synaptic recognition code. We utilized surface plasmon resonance to measure binding affinities between all combinations of alternatively spliced {beta}-NRX 1-3 and NL 1-3 ectodomains. Binding was observed for all {beta}-NRX/NL pairs. The presence of the NL1 B splice insertion lowers {beta}-NRX binding affinity by 2-fold, while {beta}-NRX splice insertion 4 has small effects that do not synergize with NL splicing. New structures of glycosylated {beta}-NRXs 1 and 2 containing splice insertion 4 reveal that the insertion forms a new {beta} strand that replaces the {beta}10 strand, leaving the NL binding site intact. This helps to explain the limited effect of splice insert 4 on NRX/NL binding affinities. These results provide new structural insights and quantitative binding information to help determine whether and how splice isoform choice plays a role in {beta}-NRX/NL-mediated synaptic recognition.

  2. Evolutionarily conserved coupling of transcription and alternative splicing in the EPB41 (protein 4.1R) and EPB41L3 (protein 4.1B) genes.

    PubMed

    Tan, Jeff S; Mohandas, Narla; Conboy, John G

    2005-12-01

    Recent studies have shown that transcription and alternative splicing can be mechanistically coupled. In the EPB41 (protein 4.1R) and EPB41L3 (protein 4.1B) genes, we showed previously that promoter/alternative first exon choice is coupled to downstream splicing events in exon 2. Here we demonstrate that this coupling is conserved among several vertebrate classes from fish to mammals. The EPB41 and EPB41L3 genes from fish, bird, amphibian, and mammal genomes exhibit shared features including alternative first exons and differential splice acceptors in exon 2. In all cases, the 5'-most exon (exon 1A) splices exclusively to a weaker internal acceptor site in exon 2, skipping a fragment designated as exon 2'. Conversely, alternative first exons 1B and 1C always splice to the stronger first acceptor site, retaining exon 2'. These correlations are independent of cell type or species of origin. Since exon 2' contains a translation initiation site, splice variants generate protein isoforms with distinct N-termini. We propose that these genes represent a physiologically relevant model system for mechanistic analysis of transcription-coupled alternative splicing. PMID:16242908

  3. A mutation in the Srrm4 gene causes alternative splicing defects and deafness in the Bronx waltzer mouse.

    PubMed

    Nakano, Yoko; Jahan, Israt; Bonde, Gregory; Sun, Xingshen; Hildebrand, Michael S; Engelhardt, John F; Smith, Richard J H; Cornell, Robert A; Fritzsch, Bernd; Bánfi, Botond

    2012-01-01

    Sensory hair cells are essential for hearing and balance. Their development from epithelial precursors has been extensively characterized with respect to transcriptional regulation, but not in terms of posttranscriptional influences. Here we report on the identification and functional characterization of an alternative-splicing regulator whose inactivation is responsible for defective hair-cell development, deafness, and impaired balance in the spontaneous mutant Bronx waltzer (bv) mouse. We used positional cloning and transgenic rescue to locate the bv mutation to the splicing factor-encoding gene Ser/Arg repetitive matrix 4 (Srrm4). Transcriptome-wide analysis of pre-mRNA splicing in the sensory patches of embryonic inner ears revealed that specific alternative exons were skipped at abnormally high rates in the bv mice. Minigene experiments in a heterologous expression system confirmed that these skipped exons require Srrm4 for inclusion into the mature mRNA. Sequence analysis and mutagenesis experiments showed that the affected transcripts share a novel motif that is necessary for the Srrm4-dependent alternative splicing. Functional annotations and protein-protein interaction data indicated that the encoded proteins cluster in the secretion and neurotransmission pathways. In addition, the splicing of a few transcriptional regulators was found to be Srrm4 dependent, and several of the genes known to be targeted by these regulators were expressed at reduced levels in the bv mice. Although Srrm4 expression was detected in neural tissues as well as hair cells, analyses of the bv mouse cerebellum and neocortex fa